Genetically optimised microorganism for producing molecules of interest

ABSTRACT

The invention concerns a genetically modified microorganism expressing a functional type I or II RuBisCO enzyme and a functional phosphoribulokinase (PRK), and in which the glycolysis pathway is at least partially inhibited, said microorganism being genetically modified so as to produce an exogenous molecule and/or to overproduce an endogenous molecule. According to the invention, the oxidative branch of the pentose phosphate pathway may also be at least partially inhibited. The invention also concerns the use of such a genetically modified microorganism for the production or overproduction of a molecule of interest and processes for the synthesis or bioconversion of molecules of interest.

FIELD OF THE INVENTION

The invention concerns a genetically modified microorganism, capable ofusing carbon dioxide as an at least partial carbon source, for theproduction of molecules of interest. More specifically, the inventionrelates to a microorganism in which at least the glycolysis pathway isat least partially inhibited. The invention also relates to processesfor the production of at least one molecule of interest using such amicroorganism.

STATE OF THE ART

Over the past few years, a number of microbiological processes have beendeveloped to enable the production of molecules of interest in largequantities.

For example, fermentation processes are used to produce molecules by amicroorganism from a fermentable carbon source, such as glucose.

Bioconversion processes have also been developed to allow amicroorganism to convert a co-substrate, not assimilable by saidmicroorganism, into a molecule of interest. Here again, a carbon sourceis required, not for the actual production of the molecule of interest,but for the production of cofactors, and more particularly NADPH, thatmay be necessary for bioconversion. In general, the production yield ofsuch microbiological processes is low, mainly due to the need forcofactors and the difficulty of balancing redox metabolic reactions.There is also the problem of the cost price of such molecules, since asource of carbon assimilable by the microorganism is still necessary. Inother words, currently, in order to produce a molecule of interest witha microbiological process, it is necessary to provide a molecule(glucose, or other), certainly of lower industrial value, but which issufficient to make the production of certain molecules not economicallyattractive.

At the same time, carbon dioxide (CO₂), whose emissions into theatmosphere are constantly increasing, is used little, if at all, incurrent microbiological processes, while its consumption bymicroorganisms for the production of molecules of interest would notonly reduce production costs, but also address certain ecologicalissues.

There is therefore still a need for microbiological processes to enablethe production of molecules of interest in large quantities and withlower cost prices than with current processes.

SUMMARY OF THE INVENTION

The advantage of using non-photosynthetic microorganisms geneticallymodified to capture CO₂ and use it as the main carbon source, in thesame way as plants and photosynthetic microorganisms, has already beendemonstrated. For example, microorganisms modified to express afunctional RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase—EC4.1.1.39) and a functional PRK (phosphoribulokinase—EC 2.7.1.19) toreproduce a partial Calvin cycle and convert ribulose-5-phosphate intotwo 3-phosphoglycerate molecules by capturing a carbon dioxide moleculehave been developed.

By working on the solutions provided by the Calvin cycle to producemolecules of interest using CO₂ as carbon source, the inventorsdiscovered that it is possible to increase the production yield ofmolecules of interest by coupling part of the Calvin cycle (PRK/RuBisCO)to at least partial inhibition of glycolysis. The inventors have alsodiscovered that it is possible to increase the consumption of exogenousCO₂ during the production of molecules of interest, by also at leastpartially inhibiting the oxidative branch of the pentose phosphatepathway. The microorganisms thus developed make it possible to produceon a large scale and with an industrially attractive yield a largenumber of molecules of interest, such as amino acids, organic acids,terpenes, terpenoids, peptides, fatty acids, polyols, etc.

The invention thus relates to a genetically modified microorganismexpressing a functional RuBisCO enzyme and a functionalphosphoribulokinase (PRK), and in which the glycolysis pathway is atleast partially inhibited, said microorganism being genetically modifiedso as to produce an exogenous molecule of interest and/or to overproducean endogenous molecule of interest, other than a RuBisCO orphosphoribulokinase enzyme.

In one particular embodiment, the genetically modified microorganism hasan oxidative branch of the pentose phosphate pathway that is also atleast partially inhibited.

The invention also concerns the use of a genetically modifiedmicroorganism according to the invention, for the production oroverproduction of a molecule of interest, preferentially selected fromamino acids, peptides, proteins, vitamins, sterols, flavonoids,terpenes, terpenoids, fatty acids, polyols and organic acids.

The present invention also concerns a biotechnological process forproducing or overproducing at least one molecule of interest,characterized in that it comprises a step of culturing a geneticallymodified microorganism according to the invention, under conditionsallowing the synthesis or bioconversion, by said microorganism, of saidmolecule of interest, and optionally a step of recovering and/orpurifying said molecule of interest.

It also concerns a process for producing a molecule of interestcomprising (i) inserting at least one sequence encoding an enzymeinvolved in the synthesis or bioconversion of said molecule of interestinto a recombinant microorganism according to the invention, (ii)culturing said microorganism under conditions allowing the expression ofsaid enzyme and optionally (iii) recovering and/or purifying saidmolecule of interest.

DESCRIPTION OF THE FIGURES

FIG. 1: General diagram of glycolysis, the pentose phosphate pathway andthe Entner-Doudoroff pathway;

FIG. 2: Schematic representation of inhibition of the glycolysispathway, according to the invention;

FIG. 3: Schematic representation of inhibition of the glycolysispathway, combined with inhibition of the oxidative branch of the pentosephosphate pathway, according to the invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “recombinant microorganism”, “modified microorganism” and“recombinant host cell” are used herein interchangeably and refer tomicroorganisms that have been genetically modified to express oroverexpress endogenous nucleotide sequences, to express heterologousnucleotide sequences, or that have an altered expression of anendogenous gene. “Alteration” means that the expression of the gene, orlevel of an RNA molecule or equivalent RNA molecules encoding one ormore polypeptides or polypeptide subunits, or the activity of one ormore polypeptides or polypeptide subunits is regulated, so that theexpression, the level or the activity is higher or lower than thatobserved in the absence of modification.

It is understood that the terms “recombinant microorganism”, “modifiedmicroorganism” and “recombinant host cell” refer not only to theparticular recombinant microorganism but to the progeny or the potentialprogeny of such a microorganism. As some modifications may occur insubsequent generations, due to mutation or environmental influences,these offspring may not be identical to the mother cell, but they arestill understood within the scope of the term as used here.

In the context of the invention, an at least partially “inhibited” or“inactivated” metabolic pathway refers to an altered metabolic pathwaythat can no longer function properly in the microorganism considered,compared with the same wild-type microorganism (not genetically modifiedto inhibit said metabolic pathway). In particular, the metabolic pathwaymay be interrupted, leading to the accumulation of an intermediatemetabolite. Such an interruption may be achieved, for example, byinhibiting the enzyme necessary for the degradation of an intermediatemetabolite of the metabolic pathway considered and/or by inhibiting theexpression of the gene encoding that enzyme. The metabolic pathway mayalso be attenuated, i.e. slowed down. Such attenuation may be achieved,for example, by partially inhibiting one or more enzymes involved in themetabolic pathway considered and/or partially inhibiting the expressionof a gene encoding at least one of these enzymes and/or by exploitingthe cofactors required for certain reactions. The expression “at leastpartially inhibited metabolic pathway” means that the level of themetabolic pathway considered is reduced by at least 20%, morepreferentially at least 30%, 40%, 50%, or more, compared with the levelin a wild-type microorganism. The reduction may be greater, and inparticular be at least greater than 60%, 70%, 80%, 90%. According to theinvention, inhibition may be total, in the sense that the metabolicpathway considered is no longer used at all by said microorganism.According to the invention, such inhibition may be temporary orpermanent.

According to the invention, “inhibition of gene expression” means thatthe gene is no longer expressed in the microorganism considered or thatits expression is reduced, compared with wild-type microorganisms (notgenetically modified to inhibit gene expression), leading to the absenceof production of the corresponding protein or to a significant decreasein its production, and in particular to a decrease of more than 20%,more preferentially 30%, 40%, 50%, 60%, 70%, 80%, 90%. In oneembodiment, inhibition can be total, i.e. the protein encoded by saidgene is no longer produced at all. Inhibition of gene expression can beachieved by deletion, mutation, insertion and/or substitution of one ormore nucleotides in the gene considered. Preferentially, inhibition ofgene expression is achieved by total deletion of the correspondingnucleotide sequence. According to the invention, any method of geneinhibition, known per se by the skilled person and applicable to amicroorganism, may be used. For example, inhibition of gene expressioncan be achieved by homologous recombination (Datsenko et al., Proc NatlAcad Sci USA. 2000; 97:6640-5; Lodish et al., Molecular Cell Biology4^(th) ed. 2000. W. H. Freeman and Company. ISBN 0-7167-3136-3); randomor directed mutagenesis to modify gene expression and/or encoded proteinactivity (Thomas et al., Cell. 1987; 51:503-12); modification of apromoter sequence of the gene to alter its expression (Kaufmann et al.,Methods Mol Biol. 2011; 765:275-94. doi: 10.1007/978-1-61779-197-0_16);targeting induced local lesions in genomes (TILLING); conjugation, etc.Another particular approach is gene inactivation by insertion of aforeign sequence, for example by transposon mutagenesis using mobilegenetic elements (transposons), of natural or artificial origin.According to another preferred embodiment, inhibition of gene expressionis achieved by knock-out techniques. Inhibition of gene expression canalso be achieved by extinguishing the gene using interfering, ribozymeor antisense RNA (Daneholt, 2006. Nobel Prize in Physiology orMedicine). In the context of the present invention, the term“interfering RNA” or “iRNA” refers to any iRNA molecule (for examplesingle-stranded RNA or double-stranded RNA) that can block theexpression of a target gene and/or facilitate the degradation of thecorresponding mRNA. Gene inhibition can also be achieved by genomeediting methods that allow direct genetic modification of a givengenome, through the use of zinc finger nucleases (Kim et al., PNAS; 93:1156-1160), transcription activator-like effector nucleases, or “TALEN”(Ousterout et al., Methods Mol Biol. 2016; 1338:27-42. doi:10.1007/978-1-4939-2932-0_3), a system combining Cas9 nucleases withclustered regularly interspaced short palindromic repeats, or “CRISPR”(Mali et al., Nat Methods. 2013 October; 10(10):957-63. doi:10.1038/nmeth.2649), or meganucleases (Daboussi et al., Nucleic AcidsRes. 2012. 40:6367-79). Inhibition of gene expression can also beachieved by inactivating the protein encoded by said gene.

In the context of the invention, “NADPH-dependent” or “NADPH-consuming”biosynthesis or bioconversion means all biosynthesis or bioconversionpathways in which one or more enzymes require the concomitant supply ofelectrons obtained by the oxidation of an NADPH cofactor.“NADPH-dependent” biosynthesis or bioconversion pathways notably concernthe synthesis of amino acids (e.g. arginine, lysine, methionine,threonine, proline, glutamate, homoserine, isoleucine, valine)γ-aminobutyric acid, terpenoids and terpenes (e.g. farnesene), vitaminsand precursors (e.g. pantoate, pantothenate, transneurosporene,phylloquinone, tocopherols), sterols (e.g. squalene, cholesterol,testosterone, progesterone, cortisone), flavonoids (e.g. frambinone,vestinone), organic acids (e.g. citric acid, succinic acid, oxalic acid,itaconic acid, coumaric acid, 3-hydroxypropionic acid), polyols (e.g.sorbitol, xylitol, glycerol), polyamines (e.g. spermidine), aromaticmolecules from stereospecific hydroxylation, via an NADP-dependentcytochrome p450 (e.g. phenylpropanoids, terpenes, lipids, tannins,fragrances, hormones).

The term “exogenous” as used here in reference to various molecules(nucleotide sequences, peptides, enzymes, etc.) refers to molecules thatare not normally or naturally found in and/or produced by themicroorganism considered. Conversely, the term “endogenous” or “native”refers to various molecules (nucleotide sequences, peptides, enzymes,etc.), designating molecules that are normally or naturally found inand/or produced by the microorganism considered.

Microorganisms

The invention proposes genetically modified microorganisms for theproduction of a molecule of interest, endogenous or exogenous.

“Genetically modified” microorganism means that the genome of themicroorganism has been modified to incorporate a nucleic sequenceencoding an enzyme involved in the biosynthesis or bioconversion pathwayof a molecule of interest, or encoding a biologically active fragmentthereof. Said nucleic sequence may have been introduced into the genomeof said microorganism or one of its ancestors, by any suitable molecularcloning method. In the context of the invention, the genome of themicroorganism refers to all genetic material contained in themicroorganism, including extrachromosomal genetic material contained,for example, in plasmids, episomes, synthetic chromosomes, etc. Theintroduced nucleic sequence may be a heterologous sequence, i.e. onethat does not naturally exist in said microorganism, or a homologoussequence. Advantageously, a transcriptional unit with the nucleicsequence of interest is introduced into the genome of the microorganism,under the control of one or more promoters. Such a transcriptional unitalso includes, advantageously, the usual sequences such astranscriptional terminators, and, if necessary, other transcriptionregulatory elements.

Promoters usable in the present invention include constitutivepromoters, i.e. promoters that are active in most cellular states andenvironmental conditions, as well as inducible promoters that areactivated or suppressed by exogenous physical or chemical stimuli, andtherefore induce a variable state of expression depending on thepresence or absence of these stimuli. For example, when themicroorganism is a yeast, it is possible to use a constitutive promoter,such as that of a gene among TEF1, TDH3, PGI1, PGK, ADH1. Examples ofinducible promoters that can be used in yeast are tetO-2, GAL10,GAL10-CYC1, PHO5.

In general, the genetically modified microorganism according to theinvention has the following features:

-   -   Expression of a functional RuBisCO (EC 4.1.1.39);    -   Expression of a functional PRK (EC 2.7.1.19);    -   At least partial inhibition of glycolysis; and    -   Expression of at least one gene involved in the synthesis and/or        bioconversion of a molecule of interest, and/or inhibition of at        least one gene encoding activity competing with the synthesis        and/or bioconversion of a molecule of interest.

According to the invention, any microorganism can be used.Preferentially the microorganism is a eukaryotic cell, preferentiallyselected from yeasts, fungi, microalgae or a prokaryotic cell,preferentially a bacterium or cyanobacterium.

In one embodiment, the genetically modified microorganism according tothe invention is a yeast, preferentially selected from among theascomycetes (Spermophthoraceae and Saccharomycetaceae), basidiomycetes(Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella) and deuteromycetes yeasts belonging to Fungi imperfecti(Sporobolomycetaceae, and Cryptococcaceae). Preferentially, thegenetically modified yeast according to the invention belongs to thegenus Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces,Candida, Lipomyces, Rhodotorula, Rhodosporidium, Yarrowia, orDebaryomyces. More preferentially, the genetically modified yeastaccording to the invention is selected from Pichia pastoris,Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae,Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis,Saccharomyces oviformis, Schizosaccharomyces pombe, Candida albicans,Candida tropicalis, Rhodotorula glutinis, Rhodosporidium toruloides,Yarrowia lipolytica, Debaryomyces hansenii and Lipomyces starkeyi.

In another embodiment, the genetically modified microorganism accordingto the invention is a fungus, and more particularly a “filamentous”fungus. In the context of the invention, “filamentous fungi” refers toall filamentous forms of subdivision Eumycotina. For example, thegenetically modified fungus according to the invention belongs to thegenus Aspergillus, Trichoderma, Neurospora, Podospora, Endothia, Mucor,Cochliobolus or Pyricularia. Preferentially, the genetically modifiedfungus according to the invention is selected from Aspergillus nidulans,Aspergillus niger, Aspergillus awomari, Aspergillus oryzae, Aspergillusterreus, Neurospora crassa, Trichoderma reesei, and Trichoderma viride.

In another embodiment, the genetically modified microorganism accordingto the invention is a microalga. In the context of the invention,“microalga” refers to all eukaryotic microscopic algae, preferentiallybelonging to the classes or superclasses Chlorophyceae, Chrysophyceae,Prymnesiophyceae, Diatomae or Bacillariophyta, Euglenophyceae,Rhodophyceae, or Trebouxiophyceae. Preferentially, the geneticallymodified microalgae according to the invention are selected fromNannochloropsis sp. (e.g. Nannochloropsis oculata, Nannochloropsisgaditana, Nannochloropsis salina), Tetraselmis sp. (e.g. Tetraselmissuecica, Tetraselmis chuii), Chlorella sp. (e.g. Chlorella salina,Chlorella protothecoides, Chlorella ellipsoidea, Chlorella emersonii,Chlorella minutissima, Chlorella pyrenoidosa, Chlorella sorokiniana,Chlorella vulgaris), Chlamydomonas sp. (e.g. Chlamydomonas reinhardtii)Dunaliella sp. (e.g. Dunaliella tertiolecta, Dunaliella salina),Phaeodactulum tricornutum, Botrycoccus braunii, Chroomonas salina,Cyclotella cryptica, Cyclotella sp., Ettlia texensis, Euglena gracilis,Gymnodinium nelsoni, Haematococcus pluvialis, Isochrysis galbana,Monoraphidium minutum, Monoraphidium sp, Neochloris oleoabundans,Nitzschia laevis, Onoraphidium sp., Pavlova lutheri, Phaeodactylumtricornutum, Porphyridium cruentum, Scenedesmus sp. (e.g. Scenedesmusobliquuus, Scenedesmus quadricaulaula, Scenedesmus sp.), Stichococcusbacillaris, Spirulina platensis, Thalassiosira sp.

In one embodiment, the genetically modified microorganism according tothe invention is a bacterium, preferentially selected from phylaAcidobacteria, Actinobacteria, Aquificae, Bacterioidetes, Chlamydia,Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres,Deinococcus-Thermus, Dictyoglomi, Fibrobacteres, Firmicutes,Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes,Proteobacteria, Spirochaetes, Thermodesulfobacteria, Thermomicrobia,Thermotogae, or Verrucomicrobia. Preferably, the genetically modifiedbacterium according to the invention belongs to the genus Acaryochloris,Acetobacter, Actinobacillus, Agrobacterium, Alicyclobacillus, Anabaena,Anacystis, Anaerobiospirillum, Aquifex, Arthrobacter, Arthrospira,Azobacter, Bacillus, Brevibacterium, Burkholderia, Chlorobium,Chromatium, Chlorobaculum, Clostridium, Corynebacterium, Cupriavidus,Cyanothece, Enterobacter, Deinococcus, Erwinia, Escherichia, Geobacter,Gloeobacter, Gluconobacter, Hydrogenobacter, Klebsiella, Lactobacillus,Lactococcus, Mannheimia, Mesorhizobium, Methylobacterium,Microbacterium, Microcystis, Nitrobacter, Nitrosomonas, Nitrospina,Nitrospira, Nostoc, Phormidium, Prochlorococcus, Pseudomonas, Ralstonia,Rhizobium, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum,Salmonella, Scenedesmun, Serratia, Shigella, Staphylococcus,Streptomyces, Synechoccus, Synechocystis, Thermosynechococcus,Trichodesmium, or Zymomonas. Also preferably, the genetically modifiedbacterium according to the invention is selected from the speciesAgrobacterium tumefaciens, Anaerobiospirillum succiniciproducens,Actinobacillus succinogenes, Aquifex aeolicus, Aquifex pyrophilus,Bacillus subtilis, Bacillus amyloliquefacines, Brevibacteriumammoniagenes, Brevibacterium immariophilum, Clostridium pasteurianum,Clostridium ljungdahlii, Clostridium acetobutylicum, Clostridiumbeigerinckii, Corynebacterium glutamicum, Cupriavidus necator,Cupriavidus metallidurans, Enterobacter sakazakii, Escherichia coli,Gluconobacter oxydans, Hydrogenobacter thermophilus, Klebsiella oxytoca,Lactococcus lactis, Lactobacillus plantarum, Mannheimiasucciniciproducens, Mesorhizobium loti, Pseudomonas aeruginosa,Pseudomonas mevalonii, Pseudomonas pudica, Pseudomonas putida,Pseudomonas fluorescens, Rhizobium etli, Rhodobacter capsulatus,Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica,Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigelladysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus,Streptomyces coelicolor, Zymomonas mobilis, Acaryochloris marina,Anabaena variabilis, Arthrospira platensis, Arthrospira maxa, Chlorobiumtepidum, Chlorobaculum sp., Cyanothece sp., Gloeobacter violaceus,Microcystis aeruginosa, Nostoc punctiforme, Prochlorococcus marinus,Synechococcus elongatus, Synechocystis sp., Thermosynechococcuselongatus, Trichodesmium erythraeum, and Rhodopseudomonas palustris.

Expression of a Functional RuBisCO and a Functional PRK

According to the invention, the microorganism can naturally express afunctional RuBisCO and a functional PRK. This is the case, for example,for photosynthetic microorganisms such as microalgae and cyanobacteria.

There are several forms of RuBisCO in nature (Tabita et al., J Exp Bot.2008; 59(7):1515-24. doi: 10.1093/jxb/erm361). Forms I, II and IIIcatalyze the carboxylation and oxygenation reactions ofribulose-1,5-biphosphate. Form I is present in eukaryotes and bacteria.It consists of two types of subunits: large subunits (RbcL) and smallsubunits (RbcS). The functional enzyme complex is a hexadecamerconsisting of eight L subunits and eight S subunits. The correctassembly of these subunits also requires the intervention of at leastone specific chaperone: RbcX (Liu et al., Nature. 2010 Jan. 14;463(7278):197-202. doi: 10.1038/nature08651). Form II is mainly found inproteobacteria, archaea (Archaea or archaebacteria) and dinoflagellatealgae. Its structure is much simpler: it is a homodimer (formed by twoidentical RbcL subunits). Depending on the organism, the genes encodinga type I RuBisCO may be called rbcL/rbcS (for example Synechococcuselongatus), or cbxLC/cbxSC, cfxLC/cfxSC, cbbL/cbbS (for exampleCupriavidus necator). Depending on the organism, the genes encoding atype II RuBisCO are generally called cbbM (for example Rhodospirillumrubrum). Form III is present in the archaea. It is generally found inthe form of dimers of the RbcL subunit, or in pentamers of dimers.Depending on the organism, the genes encoding a type III RuBisCO may becalled rbcL (for example Thermococcus kodakarensis), cbbL (for exampleHaloferax sp.).

Two classes of PRKs are known: class I enzymes found in proteobacteriaare octamers, while class II enzymes found in cyanobacteria and plantsare tetramers or dimers. Depending on the organism, the genes encoding aPRK may be called prk (for example Synechococcus elongatus), prkA (forexample Chlamydomonas reinhardtii), prkB (for example Escherichia coli),prk1, prk2 (for example Leptolyngbya sp.), cbbP (for example Nitrobactervulgaris) or cfxP (for example Cupriavidus necator).

In the case where the microorganism used does not naturally express afunctional RuBisCO and a functional PRK, said microorganism isgenetically modified to express heterologous RuBisCO and PRK.Advantageously, in such a case, the microorganism is transformed so asto integrate into its genome one or more expression cassettesintegrating the sequences encoding said proteins, and advantageously theappropriate transcription factors. Depending on the type of RuBisCO tobe expressed, it may also be necessary to have one or more chaperoneproteins expressed by the microorganism, in order to promote the properassembly of the subunits forming the RuBisCO. This is particularly thecase for type I RuBisCO, where the introduction and expression of genesencoding a specific chaperone (Rbcx) and generalist chaperones (GroESand GroEL, for example) are necessary to obtain a functional RuBisCO.Application WO2015/107496 describes in detail how to genetically modifya yeast to express a functional type I RuBisCO and PRK. It is alsopossible to refer to the method described in GUADALUPE-MEDINA et al.(Biotechnology for Biofuels, 6, 125, 2013).

In one embodiment, the microorganism is genetically modified to expressa type I RuBisCO. In another embodiment, the microorganism isgenetically modified to express a type II RuBisCO. In anotherembodiment, the microorganism is genetically modified to express a typeIII RuBisCO.

Tables 1 and 2 below list, as examples, sequences encoding RuBisCO andPRK that can be used to transform a microorganism to express afunctional RuBisCO and a functional PRK.

TABLE 1 Examples of sequences encoding a RuBisCO Gene GenBank GIOrganism rbcL BAD78320.1 56685098 Synechococcus elongatus rbcSBAD78319.1 56685097 Synechococcus elongatus cbbL2 CAJ96184.1 113529837Cupriavidus necator cbbS P09658.2 6093937 Cupriavidus necator cbbMYP_427487.1 132036 Rhodospirillum rubrum cbbM Q21YM9.1 115502580Rhodoferax ferrireducens cbbM Q479W5.1 115502578 Dechloromonas aromaticarbcL O93627.5 37087684 Thermococcus kodakarensis cbbL CQR50548.1811260688 Haloferax sp. Arc-Hr

TABLE 2 Examples of sequences encoding a PRK Gene GenBank GI Organismprk BAD78757.1 56685535 Synechococcus elongatus cfXP P19923.3 125575Cupriavidus necator PRK P09559.1 125579 Spinacia oleracea cbbP P37100.1585367 Nitrobacter vulgaris

Inhibition of Glycolysis

According to the invention, the glycolysis pathway is at least partiallyinhibited, so that the microorganism is no longer able to use thismetabolic pathway normally (FIG. 1—glycolysis). In other words, themicroorganism no longer has the ability to assimilate glucose in asimilar way to a wild-type microorganism, in which the glycolysispathway has not been inhibited (independently of any other geneticmodification).

In one particular embodiment, the microorganism is genetically modifiedto inhibit, totally or partially, glycolysis downstream of theproduction of glyceraldehyde-3-phosphate (G3P).

For example, glycolysis is inhibited upstream of the production of1,3-biphospho-D-glycerate (1,3-BPG) or upstream of the production of3-phosphoglycerate (3PG).

Depending on the microorganism, the reactions involved betweenglyceraldehyde-3-phosphate (G3P) and 3-phosphoglycerate (3PG) can bemanaged (i) by two enzymes acting concomitantly,glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12, abbreviated GAPDHor more rarely G3PDH) and phosphoglycerate kinase (E.C. 2.7.2.3,abbreviated PGK), or (ii) by a single non-phosphorylating glyceraldehyde3-phosphate dehydrogenase enzyme (EC 1.2.1.9, abbreviated GAPN).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes thereversible conversion of G3P to 1,3-biphospho-D-glycerate (1,3-BPG),using the pair NAD⁺/NADH as electron donor/acceptor in the direction ofthe reaction. Depending on the organism, the genes encoding GAPDH may becalled gapA, gapB, gapC (e.g. Escherichia coli, Arabidopsis thaliana),GAPDH, GAPD, G3PD, GAPDHS (e.g. Homo sapiens), TDH1, TDH2, TDH3 (e.g.Saccharomyces cerevisiae), gap, gap2, gap3 (e.g. Mycobacterium sp.,Nostoc sp.).

Phosphoglycerate kinase (PGK) catalyzes the reversible conversion of1,3-BPG to 3PG, using the pair ATP/ADP as cofactor. Depending on theorganism, the genes encoding PGK may be called PGK, PGK1, PGK1, PGK2,PGK3, pgkA, PGKB, PGKC, cbbK, cbbKC, cbbKP (e.g. Cupriavidus necator).

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN)catalyzes the conversion of G3P to 3PG, without going through 1,3-BPG.This reaction is catalyzed in the presence of the cofactor pairNADP⁺/NADPH, which acts as an electron acceptor. Depending on theorganism, the genes encoding GAPN may be called GAPN (e.g. Bacillus sp.,Streptococcus sp.), GAPN1 (e.g. Chlamydomonas sp.).

In one particular example, the microorganism is genetically modified sothat the expression of the gene encoding glyceraldehyde 3-phosphatedehydrogenase is at least partially inhibited. Preferentially, geneexpression is completely inhibited.

Alternatively or additionally, the expression of the gene encodingphosphoglycerate kinase may also be at least partially inhibited.Preferentially, gene expression is completely inhibited.

Alternatively, the microorganism is genetically modified so that theexpression of the gene encoding non-phosphorylatingglyceraldehyde-3-phosphate dehydrogenase is at least partiallyinhibited. Preferentially, gene expression is completely inhibited.

Tables 3, 4 and 5 below list, as examples, the sequences encoding aglyceraldehyde 3-phosphate dehydrogenase, a phosphoglycerate kinase anda non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that canbe inhibited depending on the target microorganism. The skilled personknows which gene corresponds to the enzyme of interest to be inhibiteddepending on the microorganism.

TABLE 3 Examples of sequences encoding a GAPDH Gene GenBank GI OrganismgapA NP_416293.1 947679 Escherichia coli TDH1 NP_012483.3 398364523Saccharomyces cerevisiae TDH2 NP_012542.1 6322468 Saccharomycescerevisiae TDH3 NP_011708.3 398366083 Saccharomyces cerevisiae gapECC36949.1 378544675 Mycobacterium tuberculosis gap2 P34917.2 92090599Nostoc sp.

TABLE 4 Examples of sequences encoding a PGK Gene GenBank GI Organismpgk AKL94701.1 831186507 Clostridium aceticum PGK1 NP_009938.2 10383781Saccharomyces cerevisiae pgk BAG04189.1 166089481 Microcystis aeruginosaPGKA AAG34561.2 22711882 Dictyostelium discoideum PGKB CAJ03534.168126221 Leishmania major cbbKC AAC43444.1 976365 Cupriavidus necatorpgk CAK45271.1 4982539 Aspergillus niger pgk EAU38870.1 4354973Aspergillus terreus

TABLE 5 Examples of sequences encoding a GAPN Gene GenBank GI OrganismgapN CUB58597.1 924094571 Bacillus subtilis GAPN NP_358622.1 933338Streptococcus pneumoniae GAPN1 EDP03116.1 542583 Chlamydomonasreinhardtii

In general, the production of 3-phosphoglycerate (3PG) is no longerpossible through glycolysis, or at least significantly reduced, in thegenetically modified microorganism according to the invention.

In a particular exemplary embodiment, the microorganism is a yeast ofthe genus Saccharomyces cerevisiae in which the expression of the TDH1(Gene ID: 853395), TDH2 (Gene ID: 853465) and/or TDH3 gene (Gene ID:853106) is at least partially inhibited.

In another particular exemplary embodiment, the microorganism is a yeastof the genus Saccharomyces cerevisiae in which the expression of thePGK1 gene (Gene ID: 5230) is at least partially inhibited.

In another exemplary embodiment, the microorganism is a yeast of thegenus Saccharomyces cerevisiae in which the expression of the PGK1 gene(Gene ID: 5230), the expression of the TDH1 gene (Gene ID: 853395), TDH2(Gene ID: 853465) and/or the expression of the TDH3 gene (Gene ID:853106) are at least partially inhibited.

In a particular exemplary embodiment, the microorganism is anEscherichia coli bacterium in which the expression of the gapA gene(Gene ID: 947679) is at least partially inhibited.

In another particular exemplary embodiment, the microorganism is anEscherichia coli bacterium in which the expression of the pgk gene (GeneID: 947414) is at least partially inhibited.

In another exemplary embodiment, the microorganism is an E. colibacterium in which the expression of the pgk gene (Gene ID: 947414),and/or the expression of the gapA gene (Gene ID: 947679) are at leastpartially inhibited.

According to the invention, the genetically modified microorganism,which expresses a functional RuBisCO and a functional PRK, is on theother hand capable of producing 3PG by capturing CO₂ fromribulose-5-phosphate produced by the pentose phosphate pathway (FIG. 2).

Since the enzymes necessary for the metabolism of 3PG to pyruvate arenot inhibited in the microorganism, said microorganism can thenmetabolize 3PG to produce pyruvate and ATP.

Thus, the genetically modified microorganism is able to produce pyruvateand NADPH cofactors using CO₂ as complementary carbon source.

In the context of the invention, “complementary” carbon source meansthat the microorganism uses CO₂ as a partial carbon source, in additionto the carbon atoms provided by fermentable sugars (glucose, galactose,sucrose, fructose, etc.), which constitute the majority or main carbonsource for pyruvate production.

Thus, the genetically modified microorganism according to the inventionmakes it possible to increase carbon yield, by fixing and using the CO₂normally lost during glucose metabolism via the pentose phosphatepathway, for the production of pyruvate (and subsequently molecules ofinterest).

Inhibition of the Oxidative Branch of the Pentose Phosphate Pathway

In one particular embodiment, the genetically modified microorganismaccording to the invention is also modified in such a way that theoxidative branch of the pentose phosphate pathway is also at leastpartially inhibited.

Preferentially, the microorganism is genetically modified to inhibit theoxidative branch of the pentose phosphate pathway upstream ofribulose-5-phosphate production (FIG. 1—pentose phosphate pathway).

The interruption of the oxidative branch of the pentose phosphatepathway upstream of ribulose-5-phosphate (Ru5P) production specificallytargets one or more reactions in the Ru5P synthesis process fromglucose-6-phosphate (G6P). This synthesis is generally catalyzed by thesuccessive actions of three enzymes: (i) glucose-6-phosphatedehydrogenase (EC. 1.1.1.49, abbreviated G6PDH), (ii)6-phosphogluconolactonase (E.C. 3.1.1.31, abbreviated PGL), and (iii)6-phosphogluconate dehydrogenase (EC 1.1.1.44, abbreviated PGD).

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first reactionof the pentose phosphate pathway, i.e. the oxidation ofglucose-6-phosphate to 6-phosphogluconolactone (6PGL), with concomitantreduction of one molecule of NADP to NADPH. Depending on the organism,the genes encoding G6PDH may be called G6PD (for example in Homosapiens), G6pdx (for example in Musculus), gsdA (for example inAspergillus nidulans), zwf (for example in Escherichia coli), or ZWF1(for example in Saccharomyces cerevisiae).

6-Phosphogluconolactonase (PGL) is a hydrolase that catalyzes thesynthesis of 6-phosphogluconate (6PGA) from 6PGL. Depending on theorganism, the genes encoding PGL may be called pgl (for example inEscherichia coli, Synechocystis sp.) pgls (for example inRhodobacteraceae bacterium), or SOL (for example in Saccharomycescerevisiae).

6-Phosphogluconate dehydrogenase (PGD) is an oxidoreductase thatcatalyzes the synthesis of Ru5P from 6PGA, with concomitant reduction ofan NADP molecule to NADPH and emission of a CO₂ molecule. Depending onthe organism, the genes encoding PGD may be called gnd (for example inEscherichia coli, Saccharomyces cerevisiae), PGD (for example in Homosapiens), gntZ (for example in Bacillus subtilis), or 6-PGDH (forexample in Lactobacillus paracollinoides).

In one particular example, the microorganism is genetically modified sothat the expression of the gene encoding glucose-6-phosphatedehydrogenase is at least partially inhibited. Preferentially, geneexpression is completely inhibited.

Alternatively or additionally, the microorganism is genetically modifiedso that the expression of the gene encoding 6-phosphogluconolactonase isat least partially inhibited. Preferentially, gene expression iscompletely inhibited.

Alternatively or additionally, the microorganism is genetically modifiedso that the expression of the gene encoding 6-phosphogluconatedehydrogenase is at least partially inhibited. Preferentially, geneexpression is completely inhibited.

Tables 6, 7 and 8 below list, as examples, the sequences encoding aglucose-6-phosphate dehydrogenase, a 6-phosphogluconolactonase and a6-phosphogluconate dehydrogenase that can be inhibited depending on thetarget microorganism. The skilled person knows which gene corresponds tothe enzyme of interest to be inhibited depending on the microorganism.

TABLE 6 Examples of sequences encoding a G6PDH Gene GenBank GI Organismzwf BAA15660.1 946370 Escherichia coli ZWF1 NP_014158.1 6324088Saccharomyces cerevisiae gsdA CAA54841.1 1523786 Aspergillus nidulansgsdA CAK37895.1 4979751 Aspergillus niger gsdA EAU38380.1 4316232Aspergillus terreus

TABLE 7 Examples of sequences encoding a PGL Gene GenBank GI Organismpgl BAA35431.1 4062334 Escherichia coli pgl BAK51770.1 339275283Synechocystis pgls KPQ07176.1 938272062 Rhodobacteraceae bacterium SOL3KZV10901.1 1023943655 Saccharomyces cerevisiae

TABLE 8 Examples of sequences encoding a PGD Gene GenBank GI Organismgnd ALI40222.1 937519736 Escherichia coli GND1 EDN62420.1 151944127Saccharomyces cerevisiae gntZ NP_391888.1 16081060 Bacillus subtilis6-PGDH WP_054711110.1 938929230 Lactobacillus paracollinoides

In general, the production of ribulose-5-phosphate (Ru5P) is no longerpossible through the pentose phosphate pathway, or at leastsignificantly reduced, in the genetically modified microorganismaccording to the invention.

In a particular exemplary embodiment, the microorganism is a yeast ofthe genus Saccharomyces cerevisiae in which the expression of the ZWF1gene is at least partially inhibited.

In one particular example, the yeast of the genus Saccharomycescerevisiae is genetically modified so that the expression of the TDH1,TDH2, TDH3 and/or PGK1 genes, and the expression of the ZWF1 gene are atleast partially inhibited.

In another particular exemplary embodiment, the microorganism is abacterium of the genus Escherichia coli in which the expression of thezwf gene is at least partially inhibited.

In one particular example, the bacterium of the genus Escherichia coliis genetically modified so that the expression of the gapA and/or pgkgenes, and the expression of the zwf gene are at least partiallyinhibited.

In another example, the microorganism is a filamentous fungus of thegenus Aspergillus, such as Aspergillus niger or Aspergillus terreus,genetically modified so that the expression of the pgk and gsdA genes ispartially inhibited.

According to the invention, the genetically modified microorganism,which expresses a functional RuBisCO and a functional PRK, and whoseglycolysis pathway and oxidative branch of the pentose phosphate pathwayare at least partially inhibited, is no longer capable of producing 3PGvia the glycolysis pathway or Ru5P via the oxidative branch of thepentose phosphate pathway. On the other hand, it is capable of producingRu5P by diverting the production of fructose-6-phosphate (F6P) and/orglyceraldehyde-3-phosphate (G3P), produced at the beginning ofglycolysis (upstream of inhibition). This production is possible thanksto the enzymes transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2),ribose-5-phosphate isomerase (EC 5.3.1.6), and ribulose-5-phosphateepimerase (EC 5.1.3.1) naturally present and active in themicroorganisms (FIG. 3).

Since the enzymes necessary for the metabolism of 3PG to pyruvate arenot inhibited in the microorganism according to the invention, saidmicroorganism can then metabolize 3PG to produce pyruvate and ATP.

Thus, the genetically modified microorganism is able to produce pyruvateby using exogenous CO₂ as complementary carbon source.

Thus, the genetically modified microorganism according to the inventionmakes it possible to increase the carbon yield, by fixing and usingexogenous CO₂, for the production of pyruvate (and subsequentlymolecules of interest). Here again, there is an increase in carbonyield.

Inhibition of the Entner-Doudoroff Pathway

In one particular embodiment, the genetically modified microorganismaccording to the invention has an Entner-Doudoroff pathway, and this isat least partially inhibited. This pathway, mainly found in bacteria(especially Gram-negative bacteria), is an alternative to glycolysis andthe pentose pathway for the production of pyruvate from glucose. Moreprecisely, this pathway connects to the pentose phosphate pathway atP-gluconate to feed glycolysis, particularly at pyruvate.

Preferentially, the microorganism is genetically modified to inhibitEntner-Doudoroff pathway reactions downstream of 6-phosphogluconateproduction. This inhibition eliminates a possible competing pathway, andensures the availability of 6-phosphogluconate as a substrate forPRK/RuBisCO engineering.

The interruption of the Entner-Doudoroff pathway downstream of6-phosphogluconate production specifically targets one or more reactionsin the pyruvate synthesis process from 6-phosphogluconate. Thissynthesis is initiated by the successive actions of two enzymes: (i)6-phosphogluconate dehydratase (“EDD”—EC. 4.2.1.12), and (ii)2-dehydro-3-deoxy-phosphogluconate aldolase (“EDA”—E.C. 4.1.2.14).

6-Phosphogluconate dehydratase catalyzes the dehydration of6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate. Depending onthe organism, the genes encoding 6-phosphogluconate dehydratase may becalled edd (GenBank NP_416365, for example, in Escherichia coli), orilvD (for example, in Mycobacterium sp.).

2-Dehydro-3-deoxy-phosphogluconate aldolase catalyzes the synthesis of apyruvate molecule and a glyceraldehyde-3-phosphate molecule from the2-keto-3-deoxy-6-phosphogluconate produced by 6-phosphogluconatedehydratase. Depending on the organism, the genes encoding2-dehydro-3-deoxy-phosphogluconate aldolase may be called eda (GenBankNP_416364, for example, in Escherichia coli), or kdgA (for example inThermoproteus tenax), or dgaF (for example in Salmonella typhimurium).

In one particular example, the microorganism is genetically modified sothat the expression of the gene encoding 6-phosphogluconate dehydrataseis at least partially inhibited. Preferentially, gene expression iscompletely inhibited.

Alternatively or additionally, the microorganism is genetically modifiedso that the expression of the gene encoding2-dehydro-3-deoxy-phosphogluconate aldolase is at least partiallyinhibited. Preferentially, gene expression is completely inhibited.

Tables 9 and 10 below list, as examples, the sequences encoding a6-phosphogluconate dehydratase and a 2-dehydro-3-deoxy-phosphogluconatealdolase that can be inhibited depending on the target microorganism.The skilled person knows which gene corresponds to the enzyme ofinterest to be inhibited depending on the microorganism.

TABLE 9 Examples of sequences encoding an EDD Gene GenBank GI Organismedd NP_416365.1 16129804 Escherichia coli ilvD CND70554.1 893638835Mycobacterium tuberculosis edd AJQ65426.1 764046652 Salmonella enterica

TABLE 10 Examples of sequences encoding an EDA Gene GenBank GI Organismeda AKF72280.1 817591701 Escherichia coli kdgA Q704D1.1 74500902Thermoproteus tenax eda O68283.2 81637643 Pseudomonas aeruginosa

In general, in this embodiment, pyruvate production is no longerpossible via the Entner-Doudoroff pathway, or at least significantlyreduced.

In a particular exemplary embodiment, the microorganism is a bacteriumof the genus Escherichia coli in which the expression of the edd gene isat least partially inhibited.

In one particular example, the bacterium of the genus Escherichia coliis genetically modified so that the expression of the gapA, and eddgenes are at least partially inhibited.

According to the invention, the genetically modified microorganism,which expresses a functional RuBisCO and a functional PRK, and whoseglycolysis pathway and Entner-Doudoroff pathway are at least partiallyinhibited, is no longer capable of producing 3PG by glycolysis orpyruvate by the Entner-Doudoroff pathway. The carbon flow from glucoseis therefore preferably directed towards PRK/RuBisCO engineering.

Production of Molecules of Interest

According to the invention, the genetically modified microorganism istransformed so as to produce an exogenous molecule of interest and/or tooverproduce an endogenous molecule of interest.

In the context of the invention, molecule of interest preferentiallyrefers to a small organic molecule with a molecular mass less than orequal to 0.8 kDa.

In general, genetic modifications made to the microorganism, asdescribed above, improve the carbon yield of the synthesis and/orbioconversion pathways of molecules of interest.

In the context of the invention, “improved” yield refers to the quantityof the finished product. In general, in the context of the invention,the carbon yield corresponds to the ratio of quantity of finishedproduct to quantity of fermentable sugar, particularly by weight.According to the invention, the carbon yield is increased in thegenetically modified microorganisms according to the invention, comparedwith wild-type microorganisms, placed under identical cultureconditions. Advantageously, the carbon yield is increased by 2%, 5%,10%, 15%, 18%, 20%, or more. The genetically modified microorganismaccording to the invention may produce a larger quantity of molecules ofinterest (finished product) than heterologous molecules produced by agenetically modified microorganism simply to produce or overproduce thatmolecule. According to the invention, the genetically microorganism mayalso overproduce an endogenous molecule compared with the wild-typemicroorganism. The overproduction of an endogenous molecule is mainlyunderstood in terms of quantities. Advantageously, the geneticallymodified microorganism produces at least 20%, 30%, 40%, 50%, or more byweight of the endogenous molecule than the wild-type microorganism.Advantageously, the microorganism according to the invention isgenetically modified so as to produce or overproduce at least onemolecule among amino acids, terpenoids, terpenes, vitamins and/orvitamin precursors, sterols, flavonoids, organic acids, polyols,polyamines, aromatic molecules obtained from stereospecifichydroxylation, via an NADP-dependent cytochrome p450, etc.

In one particular example, the microorganism is genetically modified tooverproduce at least one amino acid, preferentially selected fromarginine, lysine, methionine, threonine, proline, glutamate, homoserine,isoleucine, valine, and γ-aminobutyric acid.

In one particular example, the microorganism is genetically modified toproduce or overproduce molecules from the terpenoid pathway, such asfarnesene, and from the terpene pathway.

In one particular example, the microorganism is genetically modified toproduce or overproduce a vitamin or precursor, preferentially selectedfrom pantoate, pantothenate, transneurosporene, phylloquinone andtocopherols.

In one particular example, the microorganism is genetically modified toproduce or overproduce a sterol, preferentially selected from squalene,cholesterol, testosterone, progesterone and cortisone.

In one particular example, the microorganism is genetically modified toproduce or overproduce a flavonoid, preferentially selected fromframbinone and vestinone.

In one particular example, the microorganism is genetically modified toproduce or overproduce an organic acid, preferentially selected fromcoumaric acid, 3-hydroxypropionic acid, citric acid, oxalic acid,succinic acid, and itaconic acid.

In one particular example, the microorganism is genetically modified toproduce or overproduce a polyol, preferentially selected from sorbitol,xylitol and glycerol.

In one particular example, the microorganism is genetically modified toproduce or overproduce a polyamine, preferentially spermidine.

In one particular example, the microorganism is genetically modified toproduce or overproduce an aromatic molecule from a stereospecifichydroxylation, via an NADP-dependent cytochrome p450, preferentiallyselected from phenylpropanoids, terpenes, lipids, tannins, fragrances,hormones.

In the case where the molecule of interest is obtained by bioconversion,the genetically modified microorganism is advantageously cultured in aculture medium including the substrate to be converted. In general, theproduction or overproduction of a molecule of interest by a geneticallymodified microorganism according to the invention is obtained byculturing said microorganism in an appropriate culture medium known tothe skilled person.

The term “appropriate culture medium” generally refers to a sterileculture medium providing essential or beneficial nutrients for themaintenance and/or growth of said microorganism, such as carbon sources;nitrogen sources such as ammonium sulfate; sources of phosphors, forexample, potassium phosphate monobasic; trace elements, for example,salts of copper, iodide, iron, magnesium, zinc or molybdate; vitaminsand other growth factors such as amino acids or other growth promoters.An antifoam agent can be added as needed. According to the invention,this appropriate culture medium may be chemically defined or complex.The culture medium may thus be identical or similar in composition to asynthetic medium, as defined by Verduyn et al. (Yeast. 1992. 8:501-17),adapted by Visser et al. (Biotechnology and bioengineering. 2002.79:674-81), or commercially available such as yeast nitrogen base (YNB)medium (MP Biomedicals or Sigma-Aldrich).

In particular, the culture medium may include a simple carbon source,such as glucose, galactose, sucrose, molasses, or the by-products ofthese sugars, optionally supplemented with CO₂ as carbon co-substrate.According to the present invention, the simple carbon source must allowthe normal growth of the microorganism of interest. It is also possible,in some cases, to use a complex carbon source, such as lignocellulosicbiomass, rice straw, or starch. The use of a complex carbon sourceusually requires pretreatment before use.

In one particular embodiment, the culture medium contains at least onecarbon source among monosaccharides such as glucose, xylose orarabinose, disaccharides such as sucrose, organic acids such as acetate,butyrate, propionate or valerate to promote different kinds ofpolyhydroxyalkanoate (PHA), treated or untreated glycerol.

Depending on the molecules to be produced and/or overproduced, it ispossible to exploit the supply of nutritional factors (N, O, P, S, K,Mg, Fe, Mn, Co, Cu, Ca, Sn; Koller et al., Microbiology Monographs,G.-Q. Chen, 14: 85-119, (2010)). This is particularly the case topromote the synthesis and intracellular accumulation ofpolyhydroalkanoate (PHA) including polyhydroxybutyrate (PHB).

According to the invention, any culture method allowing the productionon an industrial scale of molecules of interest can be considered.Advantageously, the culture is done in bioreactors, especially in batch,fed-batch and/or continuous culture mode. Preferentially, the cultureassociated with the production of the molecule of interest is infed-batch mode corresponding to a controlled supply of one or moresubstrates, for example by adding a concentrated glucose solution whoseconcentration can be between 200 g/L and 700 g/L. A controlled supply ofvitamins during the process can also be beneficial to productivity(Alfenore et al., Appl Microbiol Biotechnol. 2002. 60:67-72). It is alsopossible to add an ammonium salt solution to limit the nitrogen supply.

Fermentation is generally carried out in bioreactors, with possiblesteps of solid and/or liquid precultures in Erlenmeyer flasks, with anappropriate culture medium containing at least a simple carbon sourceand/or an exogenous CO₂ supply, necessary for the production of themolecule of interest.

In general, the culture conditions of the microorganisms according tothe invention are easily adaptable by the skilled person, depending onthe microorganism and/or the molecule to be produced/overproduced. Forexample, the culture temperature is between 20° C. and 40° C. foryeasts, preferably between 28° C. and 35° C., and more particularlyaround 30° C., for S. cerevisiae. The culture temperature is between 25°C. and 35° C., preferably 30° C., for Cupriavidus necator.

The invention therefore also relates to the use a genetically modifiedmicroorganism according to the invention, for the production oroverproduction of a molecule of interest, preferentially selected fromamino acids, peptides, proteins, vitamins, sterols, flavonoids,terpenes, terpenoids, fatty acids, polyols and organic acids.

The invention also relates to a biotechnological process for producingat least one molecule of interest, characterized in that it comprises astep of culturing a genetically modified microorganism according to theinvention, under conditions allowing the synthesis or bioconversion, bysaid microorganism, of said molecule of interest, and optionally a stepof recovering and/or purifying said molecule of interest.

In one particular embodiment, the microorganism is genetically modifiedto express at least one enzyme involved in the synthesis of saidmolecule of interest.

In another particular embodiment, the microorganism is geneticallymodified to express at least one enzyme involved in the bioconversion ofsaid molecule of interest.

The invention also relates to a process for producing a molecule ofinterest comprising (i) inserting at least one sequence encoding anenzyme involved in the synthesis or bioconversion of said molecule ofinterest into a recombinant microorganism according to the invention,(ii) culturing said microorganism under conditions allowing theexpression of said enzyme and optionally (iii) recovering and/orpurifying said molecule of interest.

For example, it is possible to overproduce citrate by a fungus,particularly a filamentous fungus, such as Aspergillus niger,genetically modified to express a functional PRK and a functional type Ior II RuBisCO, and in which the expression of the pgk (Gene ID: 4982539)and gsdA (Gene ID: 497979751) genes is at least partially inhibited.

It is also possible to overproduce itaconic acid by a fungus,particularly a filamentous fungus, such as Aspergillus terreus orAspergillus niger, genetically modified to express a functional PRK anda functional type I or II RuBisCO, and in which the expression of thepgk (Gene ID: 4354973) and gsdA (Gene ID: 4316232) genes is at leastpartially inhibited.

Similarly, it is possible to produce farnesene by a yeast such as ayeast of the genus Saccharomyces cerevisiae genetically modified toexpress a functional PRK and a functional type I or II RuBisCO, afarnesene synthase and in which the expression of a PGK1 gene (Gene ID:5230) is at least partially inhibited.

It is also possible to overproduce glutamate by a bacterium, such as abacterium of the genus Escherichia coli, genetically modified to expressa functional PRK and a functional type I or II RuBisCO, and in which theexpression of the gapA gene (Gene ID: 947679) is at least partiallyinhibited. This overproduction can also occur in a strain where at leastpartial inhibition of the gapA gene is combined with at least partialinhibition of the zwf gene (Gene ID: 946370).

Similarly, it is also possible to overproduce γ-aminobutyric acid by abacterium, such as a bacterium of the genus Escherichia coli,genetically modified to express a functional PRK and a functional type Ior II RuBisCO, as well as a glutamate decarboxylase gadB (Gene ID:946058), and in which the expression of the gapA gene (Gene ID: 947679)is at least partially inhibited. This overproduction can also occur in astrain where at least partial inhibition of the gapA gene is combinedwith at least partial inhibition of the zwf gene (Gene ID: 946370).

Similarly, it is possible to overproduce succinic acid and oxalic acidby a bacterium, such as a bacterium of the genus Escherichia coli,genetically modified to express a functional PRK and a functional type Ior II RuBisCO, as well as an enzymatic activity allowing the oxidationof glyoxylate to oxalate, preferentially a glyoxylate dehydrogenaseFPGLOXDH1 (mRNA: BAH29964.1), a glyoxylate oxidase GLO (mRNA:AOW73106.1), or a lactate dehydrogenase LDHA (Gene ID: 3939), and inwhich the expression of the gapA (Gene ID: 947679) and zwf (Gene ID:946370) genes is at least partially inhibited.

EXAMPLES Example 1: Bioinformatics Analysis

a) Calculation of Theoretical Yields

i) Comparison of Carbon Fixation Yields from Glucose Between a Wild-TypeStrain Using the Pentose Phosphate Pathway and Glycolysis and a ModifiedStrain According to the Invention

In order to evaluate the benefit of the modifications describedaccording to the invention, theoretical yield calculations were carriedout on the basis of the stoichiometry of the reactions involved.

Two scenarios were analyzed: the improvement provided by PRK-RuBisCOengineering (i) in a strain inhibited for glycolysis on the yield of aNADPH-dependent biosynthetic pathway (for example farnesene synthesis),and (ii) in a strain inhibited for glycolysis and for the oxidativebranch of the pentose phosphate pathway on the yield of a biosyntheticpathway of interest (for example citrate synthesis).

In the context of the improvement of NADPH-dependent biosyntheticpathways, the theoretical balance of the formation of NADPH andglyceraldehyde-3-phosphate (G3-P) from glucose via the pentose phosphatepathway was calculated according to the following equation (1):

3Glucose+5ATP+6NADP⁺+3H₂O→5G3-P+5ADP+6NADPH+11H⁺+3CO₂  (1)

Going down to pyruvate formation from G3P, we arrive at the followingbalance:

3Glucose+5ADP+6NADP⁺+5NAD⁺+5P_(i)→5Pyruvate+5ATP+6NADPH+5NADH+11H⁺+3CO₂+2H₂O  (2)

If we normalize the balance for one mole of glucose, we obtain thefollowing yield:

Glucose+1.67ADP+2NADP⁺+1.67NAD⁺+1.67P_(i)→1.67Pyruvate+1.67ATP+2NADPH+1.67NADH+3.67H⁺+CO₂+0.67H₂O  (3)

Thus, by using the pentose phosphate pathway, 1.67 moles of pyruvate and2 moles of NADPH are produced from one mole of glucose. However, onemole of carbon is lost by decarboxylation when ribulose-5-phosphate isformed by 6-phosphogluconate dehydrogenase (EC 1.1.1.44). In comparison,pyruvate formation by the glycolysis pathway gives the following yield:

Glucose+2ADP+2NAD⁺+2P_(i)→2Pyruvate+2ATP+2NADH+2H⁺+2H₂O  (4)

The maximum theoretical yield of pyruvate production by the pentosephosphate pathway is therefore 0.82 g_(pyruvate)/g_(glucose) (g ofsynthesized pyruvate, per g of glucose consumed), while it is 0.98g_(pyruvate)/g_(glucose) by the glycolysis pathway.

By integrating PRK/RuBisCO engineering into a strain inhibited forglycolysis (for example ΔPGK1 in S. cerevisiae yeast), the carbonfixation flux is redirected to the oxidative branch of the pentosephosphate pathway and then to PRK/RuBisCO engineering (see FIG. 2). Thisflux is related to the end of the glycolysis pathway, at the level of3-phosphoglycerate (3PG) formation, with the following yield:

Glucose+2ATP+2NADP⁺+2H₂O→2 3PG+2ADP+2NADPH+6H⁺  (5)

Going down to pyruvate formation from 3PG, we arrive at the followingbalance:

Glucose+2NADP⁺→2Pyruvate+2NADPH+4H⁺  (6)

The integration of the modifications according to the invention into amicroorganism makes it possible to recover the carbon molecule otherwiselost by decarboxylation in the pentose pathway. The maximum theoreticalcarbon fixation yield is therefore 0.98 g_(pyruvate)/g_(glucose), whichimproves by 20.5% the yield obtained by the production of pyruvate bythe pentose phosphate pathway, while producing NADPH.

In a second case (see FIG. 3), PRK/RuBisCO engineering is integratedinto a strain that is both inhibited for glycolysis (for example ΔPGK1in the case of S. cerevisiae yeast) and for the oxidative branch of thepentose phosphate pathway (for example ΔZWF1 in the case of S.cerevisiae yeast). The theoretical balance of the formation of NADPH and3-phosphoglycerate (3PG) from glucose then becomes

2.5Glucose+6ATP+3CO₂+3H₂O→6 3PG+6ADP+12H⁺  (7)

Going down to pyruvate formation from 3PG, we arrive at the followingbalance

2.5Glucose+3CO₂→6Pyruvate++3H₂O+6H⁺  (8)

If we normalize the balance for one mole of glucose, we obtain thefollowing yield:

Glucose+1.2CO₂→2.4Pyruvate+1.2H₂O+2.4H⁺  (9)

The integration of the modifications according to the invention makes itpossible to fix 1.2 additional carbon molecule per mole of glucoseconsumed. The corresponding maximum theoretical yield is 1.17g_(pyruvate)/g_(glucose), which is ˜20% improvement compared with thecarbon fixation yield of glycolysis.

ii) Application to Citrate Production

In a second case, the calculation is applied to citrate production in S.cerevisiae yeast, in a wild-type strain and in a modified strainmodified according to the invention incorporating PRK/RuBisCOengineering and deleted for the PGK1 gene so as to inhibit theglycolysis pathway, and for the ZWF1 gene to inhibit the oxidativebranch of the pentose pathway.

The production of citrate from pyruvate is summarized by the followingbalance equation:

2Pyruvate+ATP+NAD⁺+2H₂O→Citrate+ADP+NADH+P_(i)+3H⁺  (11)

This synthesis does not require NADPH, but 2 moles of pyruvate.Optimally, a wild-type strain obtains these 2 moles of pyruvate byglycolysis, from one mole of glucose according to equation (4), with thefollowing balance:

Glucose+ADP+3NAD⁺+P_(i)→Citrate+ATP+3NADH+5H⁺  (12)

The corresponding g_(citrate)/g_(glucose) yield is 1.07

In the context of a modified strain according to the invention,inhibited for the glycolysis pathway and the pentose phosphate pathway,the 2 pyruvates required are obtained with only 0.83 mole of glucose(see equation 9), with the following balance:

0.83Glucose+CO₂+ATP+NAD⁺+H₂O→Citrate+ADP+NADH+P_(i)+5H⁺  (13)

The corresponding g_(citrate)/g_(glucose) yield is 1.28, a maximumtheoretical increase of about 20% compared with the yield of thewild-type strain.

b) Simulation of Biosynthesis Yields by Flux Balance Analysis

In a bioinformatics approach, flux balance analyses (FBAs) were alsoperformed to simulate the impact of the modifications describedaccording to the invention on the yield of different biosyntheticpathways.

FBAs are based on mathematical models that simulate metabolic networksat the genome scale (Orth et al., Nat Biotechnol. 2010; 28: 245-248).Reconstructed networks contain the known metabolic reactions of a givenorganism and integrate the needs of the cell, in particular to ensurecell maintenance or growth. FBAs make it possible to calculate the flowof metabolites through these networks, making it possible to predicttheoretical growth rates as well as metabolite production yields.

i) Procedure

FBA simulations were performed with the OptFlux software (Rocha et al.,BMC Syst Biol. 2010 Apr. 19; 4:45. doi: 10.1186/1752-0509-4-45), and theSaccharomyces cerevisiae metabolic model iMM904 (Mo et al., BMC SystBiol. 2009 Mar. 25; 3:37. doi: 10.1186/1752-0509-37). This model hasbeen modified to include the improvements described according to theinvention, including a heterologous CO₂ fixation pathway with (i) theaddition of a PRK-type reaction, (ii) the addition of a RuBisCO-typereaction.

In particular exemplary embodiments, the reactions necessary to simulatethe production of molecules through heterologous pathways have also beenadded to the model.

In a particular exemplary embodiment, a farnesene synthase reaction (EC4.2.3.46 or EC 4.2.3.47) has been added for the heterologous productionof farnesene.

In a second particular exemplary embodiment, acetoacetyl-CoA reductase(EC 1.1.1.36) and poly-hydroxybutyrate synthase (EC 2.3.1.B2 or2.3.1.B5) reactions were added to the model to simulate a heterologousproduction pathway of β-hydroxybutyrate, the monomer ofpolyhydroxybutyrate.

In another particular exemplary embodiment, a glutamate decarboxylasereaction (EC 4.1.1.15) was added for the heterologous production ofγ-aminobutyric acid.

In another particular exemplary embodiment, an aconitate decarboxylasereaction (EC 4.1.1.6) was added for the heterologous production ofitaconic acid.

In another particular exemplary embodiment, a lactate dehydrogenasereaction (EC 1.1.1.27) was added for the heterologous production ofoxalate

The simulations were carried out by applying to the model a set ofconstraints reproducible by the skilled person, aimed at simulating thein vivo culture conditions of a strain of S. cerevisiae under theconditions described according to the invention (for example presence ofunrestricted glucose in the medium, aerobic culture condition).

In particular exemplary embodiments, simulations are performed byvirtually inactivating the reactions of the enzymes PGK1 (for exampleglutamate, β-hydroxybutyric acid, farnesene) and ZWF1 (for examplecitrate production), in order to simulate the decreases in glycolysisactivity and the pentose phosphate pathway, described according to theinvention.

Simulations are carried out in parallel on an unmodified “wild-typestrain” model in order to evaluate the impact of the improvementsdescribed according to the invention on the production yield of thebiosynthetic pathways tested.

ii) Results

The theoretical yields obtained and the percentages of improvementprovided by the invention are described in Table 11 below.

TABLE 11 Maximum theoretical production yields evaluated by FBA on awild-type strain and a modified strain according to the modifications ofthe patent, for the production of different molecules. Percentageimprovement in theoretical Maximum theoretical Maximum theoreticalproduction mass production yields yields with a modified strainefficiency with a wild-type strain according to the inventiong_(x)/g_(GLUC) Target Simulation Mol_(x)/ CMol_(x)/ Mol_(x)/ CMol_(x)/provided by molecule conditions Mol_(GLUC) CMol_(GLUC) g_(x)/g_(GLUC)Mol_(GLUC) CMol_(GLUC) g_(x)/g_(GLUC) the invention Citrate ΔPGK1, 1 11.07 1.2 1.2 1.28   +20% ΔZWF1, Itaconate ΔPGK1, 1 0.83 0.72 1.2 1 0.87  +20% ΔZWF1, Glutamate ΔPGK1 0.92 0.77 0.75 1.09 0.91 0.89 +18.7%ΔPGK1, 1 0.83 0.82 1.2 1 0.98   +20% ΔZWF1, GABA ΔPGK1, 1 0.67 0.57 1.20.8 0.69   +20% ΔZWF1, β- ΔPGK1 0.92 0.61 0.53 1.09 0.73 0.63 +18.2%Hydroxybutyric acid Farnesene ΔPGK1 0.21 0.54 0.24 0.24 0.59 0.27 +12.5%Co-production Succinate ΔPGK1, 1 0.67 0.66 1.2 0.8 0.79   +20% OxalateΔZWF1, 1 0.33 0.5 1.2 0.4 0.6   +20% Mol_(x)//Mol_(GLUC): moles ofmolecule X produced, in relation to the moles of glucose consumedCMol_(x)/CMol_(GLUC):: moles of carbon of molecule X produced, inrelation to the moles of carbon of glucose consumed g_(x)/g_(GLUC): g ofmolecule X produced, in relation to the g of glucose consumed.

Example 2: Improvement of Farnesene Production in S. cerevisiae

A Saccharomyces cerevisiae yeast strain, CEN.PK 1605 (Mat a HIS3leu2-3.112 trp1-289 ura3-52 MAL.28c) derived from the commercial strainCEN.PK 113-7D (GenBank: JRIV00000000 is engineered to produce NADPHwithout CO₂ loss and thus allow the improvement of alpha-farneseneproduction from glucose.

a) Inactivation of the Glycolysis Pathway

To that end, the glycolysis pathway was inactivated by deletion of thePGK1 gene. Once glycolysis is inhibited, the resulting yeast strain isno longer able to use glucose as a source of carbon and energy. It istherefore necessary to supply the biomass synthesis pathways withglycerol and the energy pathways with ethanol. The strains in which PGK1is deleted are grown on YPGE (yeast extract peptone glycerol ethanol)medium.

The deletion of the PGK1 gene was obtained as follows:

The coding phase of the G418 resistance gene, derived from the KanMXcassette contained on plasmid pUG6 (P30114—Euroscarf), was amplifiedwith the oligonucleotides CB101 (SEQ ID NO: 1) and CB102 (SEQ ID NO: 2):

SEQ ID NO: 1: CB101 (forward):5′-ACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATATAAAACAATGGGTAAGGAAAAGACTCACGTTTC-3′ SEQ ID NO: 2: CB102 (reverse):5′-GGGAAAGAGAAAAGAAAAAAATTGATCTATCGATTTCAATTCAATTCAATTTAGAAAAACTCATCGAGCATCAAATGAAAC-3′

The underlined portion of the oligonucleotides is perfectly homologousto the Kan sequence and the rest of the sequence corresponds to theregions adjacent to the coding phase of the PGK1 gene on theSaccharomyces cerevisiae genome so as to generate a PCR ampliconcontaining at its ends homologous recombination sequences of the PGK1gene locus.

For the transformation reaction according to the skilled man (Methods inYeast Genetics, Cold Spring Harbor lab course manual, 1997; Gietz andSchiest, 1995, Methods in Molecular and Cellular Biology 5[5]:225-269),strain CEN.PK 1605 was grown in a volume of 50 mL of complex rich mediumYPD (yeast extract peptone dextrose) at 30° C. to an optical density at600 nm of 0.8. The cells were centrifuged for 5 minutes at 2,500 rpm atroom temperature.

The supernatant was removed and the cells were resuspended in 25 mL ofsterile water and centrifuged again for 5 minutes at 2,500 rpm at roomtemperature. After removing the supernatant, the cells were resuspendedin 400 μL of 100 mM sterile lithium acetate.

At the same time, a transformation mix was prepared in a 2 mL tube asfollows: 250 μL of 50% PEG, 10 μL of “carrier” DNA at 5 mg/mL, 36 μL of1 M lithium acetate, 5 or 10 μL of purified PCR reaction (deletioncassette) and 350 μL of water.

The resuspended cells (50 μL) were added to the transformation mixtureand incubated at 42° C. for 40 minutes in a water bath.

After incubation, the tube was centrifuged for 1 minute at 5,000 rpm atroom temperature and the supernatant was discarded. The cells wereresuspended in 2 mL of YPGE (yeast extract peptone glycerol ethanol)medium, transferred to a 14 mL tube and incubated for 2 hours at 30° C.at 200 rpm. The cells were then centrifuged for 1 minute at 5,000 rpm atroom temperature. The supernatant was removed and the cells wereresuspended in 1 mL of sterile water and centrifuged again for 1 minuteand resuspended in 100 μL of sterile water and spread over 180 μg/mLYPGE+G418.

The colonies obtained were genotyped for the validation of the deletionof the PGK1 gene and referenced EQ-0134 (CEN.PK1605 Δpgk1::kan).

b) Introduction of PRK—RuBisCO—Alpha-Farnesene Synthase Enzymes

In order to reconstitute an alternative pathway to glycolysis and allowthe Δpgk1 strain to grow on glucose, said strain has been modified toallow combinatorial expression of:

-   -   a gene encoding a phosphoribulokinase PRK which is grafted onto        the pentose phosphate pathway by consuming ribulose-5P to give        ribulose-1.5bisP and    -   a type I RuBisCO (with the structural genes RbcL and RbcS and        the chaperones RbcX, GroES and GroEL). RuBisCO consumes        ribulose-1.5bisP and one mole of CO₂ to form 3-phosphoglycerate        downstream of the PGK1 deletion in the glycolysis pathway.

This alternative pathway once again allows the strain to consume glucoseas its main source of carbon and energy.

To produce alpha-farnesene, the yeast lacks the alpha-farnesene synthasegene (AFS1; SEQ ID NO: 71; GenBank accession number AY182241).

Also, the seven genes required for PRK-RuBisCO engineering (Table 12)were cloned on four plasmid vectors capable of autonomous replication,with compatible origins of replication and each carrying a differentgene for complementation of auxotrophy or of antibiotic resistance,allowing the selection of strains containing the three or four plasmidconstructs.

Two of these plasmids are single-copy, with an Ars/CEN origin ofreplication and the third is multicopy with a 2μ origin.

TABLE 12 Description of expression cassettes and plasmid compositionCodon Auxo- optimi- Termi- trophic GenBank zation Promoter nator orimarker Plasmids RbcL BAD78320.1 Yes TDH3p ADH1t 2μ URA3 pFPP45 RbcSBAD78319.1 Yes TEF1p PGK1t 2μ URA3 pFPP45 RbcX BAD80711.1 Yes TEF1pPGK1t ARS-CEN6 LEU2 pFPP56 GroES U00096 No PGI1p CYC1t ARS-CEN6 LEU2pFPP56 GroEL AP009048 No TDH3p ADH1t ARS-CEN6 LEU2 pFPP56 PRK BAD78757.1Yes Tet-OFF CYC1t ARS416-CEN4 TRP1 pFPP20 alpha- AY182241 Yes TEF1pPGK1t 2μ NatMX pL4 farnesene synthase (AFS1) Empty Tet-OFF CYC1tARS416-CEN4 TRP1 pCM185 Empty ARS-CEN6 LEU2 pFL36 Empty TEF1p PGK1t 2μURA3 pV51TEF

Genes from Synechococcus elongatus such as rbcL, rbcS, rbcX and prk (asdescribed in WO 2015107496 A1) and Malus domestica alpha-farnesenesynthase (Tippmann et al., Biotechnol Bioeng. 2016 January;113(1):72-81) have been optimized for the use of codons in Saccharomycescerevisiae yeast.

According to the protocol previously described for yeast transformation,strain EQ-0134 was grown in a volume of 50 mL of complex rich mediumYPGE (yeast extract peptone glycerol ethanol) at 30° C. The cells arecentrifuged for 5 minutes at 2,500 rpm at room temperature. Thesupernatant is removed and the cells are resuspended in 25 mL of sterilewater and centrifuged again for 5 minutes at 2,500 rpm at roomtemperature. After removing the supernatant, the cells are resuspendedin 400 μL of 100 mM sterile lithium acetate. At the same time, thefollowing transformation mix is prepared: 250 μL of 50% PEG, 10 μL of“carrier” DNA at 5 mg/mL, 36 μL of 1 M lithium acetate, 10 μL (3 μg ofone of the following combinations, pFPP45+pFPP56+pFPP20 orpL4+pFPP45+pFPP56+pFPP20) and 350 μL of water.

The resuspended cells (50 μL) were added to the transformation mixtureand incubated at 42° C. for 40 minutes in a water bath. Afterincubation, the tube was centrifuged for 1 minute at 5,000 rpm at roomtemperature and the supernatant was discarded. The cells wereresuspended in 2 mL YNB (yeast nitrogen base including ammonium sulfate)with glycerol and ethanol, transferred to a 14 mL tube and incubated for2 hours at 30° C. under atmosphere enriched with 10% CO₂. The final mixis spread on YNB agar medium including ammonium sulfate+CSM without LUW(leucine uracil, tryptophan)+nourseothricin if applicable, with glyceroland ethanol as carbon sources.

According to the previously described protocol, strain CEN.PK 1605 istransformed with the following plasmid combination:pL4+pFL36+pCM185+pV51TEF.

The clones obtained were genotyped for all engineering genes and thenadapted on liquid medium YNB ammonium sulfate and glucose.

-   -   EQ-0153 (CEN.PK1605 Δpgk1::kan) (pFPP45+pFPP56+pFPP20)    -   EQ-0253 (CEN.PK1605 Δpgk1::kan) (pL4+pFPP56+pFPP20+pFPP45)    -   EQ-0353 (CEN.PK1605) (pL4+pFL36+pCM185+pV51TEF)

c) Adaptation of Strains EQ-0153 and EQ-0253 to Growth in Liquid Mediumwith Glucose and CO₂.

Batch-mode cultures in Erlenmeyer flasks are carried out with theappropriate culture medium and a 10% exogenous CO₂ supply, in a shakingincubator (120 rpm, 30° C.), with inoculation at 0.05 OD 600 nm measuredusing an EON spectrophotometer (BioTek Instruments). The strain ofinterest is grown on YNB+CSM-LUW medium with 10 g/L glycerol and 7.5 g/Lethanol, under conditions where PRK expression is not induced, and inthe presence of nourseothricin if appropriate. Under these conditions,it is necessary to feed the strain before and after the deletion of thePGK1 gene.

After obtaining a sufficient quantity of biomass, cultures with a volumegreater than or equal to 50 mL in Erlenmeyer flasks of at least 250 mLare inoculated in order to adapt the strain to the use of thePRK/RuBisCO engineering. This adaptation is carried out on YNB+CSM-LUWculture medium with 20 g/L glucose, in the presence of nourseothricin ifnecessary and an exogenous CO₂ supply as described above.

After observation of a significant growth start, the strains are adaptedto a minimum mineral medium free of the amino acids and nitrogenousbases included in the CSM-LUW, i.e. only YNB with 20 g/L glucose,nourseothricin if necessary and an exogenous CO₂ supply as describedabove.

d) Production of Farnesene in Erlenmeyer Flasks

Saccharomyces cerevisiae strain EQ-0253, with a deletion in theglycolytic pathway at the PGK1 gene, is grown to produce farnesene whileoverproducing NADPH without CO₂ loss, using a PRK and a RuBisCO.

This strain of interest is compared with a reference strain EQ-0353producing farnesene following the introduction of a heterologousalpha-farnesene synthase, without deletion of PGK1 or addition of PRKand RuBisCO.

Strains EQ-0253 (CEN.PK1605 Δpgk1::kan) (pL4+pFPP56+pFPP20+pFPP45) andEQ-0353 (CEN.PK1605) (pL4+pFL36+pCM185+pV51TEF) were grown in a YNBmedium with 20 g/L D-glucose, to which 100 μg/L nourseothricin wasadded. A pre-culture containing 20 mL of culture medium was inoculatedat 0.05 OD_(600 nm) into a 250 mL baffled Erlenmeyer flask, shaken at120 rpm for 24 h at 30° C. in a Minitron incubator with an atmosphereregulated at 10% CO₂. From the first pre-culture, 50 mL of medium wasinoculated at 0.05 OD_(600 nm) into a 250 mL Erlenmeyer and shaken at120 rpm for 24 h at 30° C., 10% CO₂. The culture, also conducted inErlenmeyer flasks (500 mL, baffled) from the second pre-culture, wasinoculated at 0.05 OD_(600 nm) into 100 mL of the same culture medium,to which 50 μg/mL ampicillin, 10 μL antifoam (Antifoam 204, Sigma,A6426) and 10% (v/v) dodecane were added (Tippman et al., Talanta(2016), 146: 100-106). The cultures were shaken at 120 rpm at 30° C. inthe presence of 10% CO₂. Growth was monitored by measuring turbidity at600 nm.

To extract farnesene, 500 μL of organic phase was collected andcentrifuged at 5,000 g for 5 min for complete separation of the twophases. The organic phase was stored at 4° C. until GC-MS analysis. Thedetection and quantification of α-farnesene was performed by singlequadrupole mass spectrometry. A Zebron ZB-FFAP column was used withhydrogen as the carrier gas at a fixed rate of 2.95 mL/min. The inlettemperature was 260° C., 1 μL of sample was injected in splitless mode.The initial oven temperature was 70° C. (4 min) then it was graduallyincreased to 160° C. (7° C./min) then to 240° C. (40° C./min) where itwas maintained for 1.05 min. For mass spectrometric detection, thetransfer line and source temperatures were 250° C. and 200° C.respectively. The mass acquisition was made between t=10 min and t=20min. An external calibration including seven points was performed usingthe farnesene isomer mix (Sigma, W383902) for the quantification ofα-farnesene produced by the strains.

To quantify the glucose consumed by the strains, 500 μL of culturemedium was collected at the same farnesene extraction OD, centrifuged at5,000 g, 5 min at 4° C. The supernatant was filtered (Minicart RC4,Sartorius 0.45 μm) and stored in a flask at −20° C. The glucosecontained in this sample was quantified by UltiMate 3000 HPLC-UV (ThermoScientific) equipped with a pump, an 8° C. refrigerated autosampler anda refractive index (RI) detector (Precision Instruments IOTA 2). A RezexROA-Organic Acid H⁺ column (8%) 150×7 8 mm, 8 μm particle size(Phenomenex, 00H-0138-KO) was used with a Carbo-H pre-column 4×3.0 mm.The temperature of the column was 35° C. and the flow rate was set at0.5 mL/min. Isocratic elution was performed with an aqueous mobile phaseat 5 mM H₂SO₄ and lasted 30 min A volume of 20 μL was injected for eachsample. The identification of compounds was based on the comparison ofretention times with standards. The external calibration includes 10points of variable glucose concentration (0-20 g/L).

The carbon yield Y_(α-farnesene/Glc) is calculated in grams of farneseneproduced per gram of glucose consumed for both strains EQ-0253 andEQ-0353,

${Y_{\alpha - {{farnesene}/{Glc}}} = \frac{{farnesene}\mspace{14mu} \left( {{mg}\text{/}L\mspace{14mu} {aqueous}} \right)}{{glucose}\mspace{14mu} \left( {{mg}\text{/}L\mspace{14mu} {aqueous}} \right)}}.$

TABLE 13 Mass yield of α-farnesene to D-glucose Y_(α-farnesene/glucose)Yield Strains (×10⁻⁴) (g/g) improvement EQ-0253 12.5 +9.6% EQ-0353 11.4

The increase in the mass yield of α-farnesene to D-glucose observed was9.6% for strain EQ-0253, compared with control strain EQ-0353.

Example 3: Improvement of Citrate Production in S. cerevisiae

a) Inactivation of the ZWF1 Gene and the IDH1 Gene in a Haploid Strainof Mating Type MAT a

Inactivation of the ZWF1 Gene

The coding phase of the hygromycin B resistance gene, derived from thehphMX cassette (loxP-pAgTEF1-hph-tAgTEF1-loxP) and contained on plasmidpUG75 (P30671)—Euroscarf), is amplified with the oligonucleotides Sdzwf1and Rdzwf1 (Table 14). This makes it possible to generate a Δzwf1 PCRamplicon containing at its ends homologous recombination sequences ofthe glucose-6-phosphate dehydrogenase ZWF1 gene locus.

TABLE 14 Oligonucleotides Name Sequence Sdzwf1AAGAGTAAATCCAATAGAATAGAAAACCACATAAGGCAAGA (SEQ IDTGGGTAAAAAGCCTGAACTCACCG NO: 3) Rdzwf1ATTTCAGTGACTTAGCCGATAAATGAATGTGCTTGCATTTT (SEQ ID TTTATTCCTTTGCCCTCGGACGNO: 4) Sdpgk1 ACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAA (SEQ IDTATAAAACAATGGGTAAGGAAAAGACTCACGTTTC NO: 5) Rdpgk1GGGAAAGAGAAAAGAAAAAAATTGATCTATCGATTTCAATT (SEQ IDCAATTCAATTTAGAAAAACTCATCGAGCATCAAATGAAAC NO: 6) Sdidh1TCTCCCTATCCTCATTCTTCTCCCTTTTCCTCCATAATTGT (SEQ IDAAGAGAAAAATGGGTACCACTCTTGACGACACGG NO: 7) Rdidh1AATTTGAACACACTTAAGTTGCAGAACAAAAAAAAGGGGAA (SEQ IDTTGTTTTCATTAGGGGCAGGGCATGCTCATGTAGAGC NO: 8)

The underlined portion of the oligonucleotides corresponds to theportion perfectly homologous to the sequence of the selection gene, therest of the sequence corresponding to the regions adjacent to the codingphase of the target gene to be deleted on the Saccharomyces cerevisiaegenome.

The previously described strain CEN.PK 1605 (Mat a HISS leu2-3.112trp1-289 ura3-52 MAL.28c) derived from the commercial strain CEN.PK113-7D (GenBank: JRIV00000000) is transformed with the Δzwf1 PCRfragment described above.

For the transformation reaction, strain CEN.PK 1605 is grown in a volumeof 50 mL of complex rich medium YPD (yeast extract peptone dextrose,here 20 g/L glucose) at 30° C. to an optical density at 600 nm of 0.8.The cells are centrifuged for 5 minutes at 2,500 rpm at roomtemperature. The supernatant is removed and the cells are resuspended in25 mL of sterile water and centrifuged again for 5 minutes at 2,500 rpmat room temperature. After removing the supernatant, the cells areresuspended in 400 μL of 100 mM sterile lithium acetate.

At the same time, a transformation mix is prepared in a 2 mL tube asfollows: 250 μL of 50% PEG, 10 μL of “carrier” DNA at 5 mg/mL, 36 μL of1 M lithium acetate, 10 μL of purified PCR reaction (deletion cassette)and 350 μL of water.

The resuspended cells (50 μL) are added to the transformation mixtureand incubated at 42° C. for 40 minutes in a water bath. Afterincubation, the tube is centrifuged for 1 minute at 5,000 rpm at roomtemperature and the supernatant is discarded. The cells are resuspendedin 2 mL of YPD (yeast extract peptone dextrose) medium, transferred to a14 mL tube and incubated for 2 hours at 30° C. at 200 rpm. The cells arethen centrifuged for 1 minute at 5,000 rpm at room temperature. Thesupernatant is removed and the cells are resuspended in 1 mL of sterilewater and centrifuged again for 1 minute and resuspended in 100 μL ofsterile water and spread on YPD+HygromycinB (200 μg/mL).

The colonies obtained were genotyped for the validation of the deletionof the ZWF1 gene and referenced EQSC-002 (CEN.PK 1605 Δzwf1::hph).

Inactivation of the IDH1 Gene

Inactivation of this gene allows citrate to accumulate (Rodriguez etal., Microb Cell Fact. 2016 Mar. 3; 15:48).

The coding phase of the nourseothricin resistance gene, derived from thenatMX cassette (loxP-pAgTEF1-nat-tAgTEF1-loxP) contained on the plasmid(pUG74 (P30670)—Euroscarf) is amplified with the oligonucleotides Sdidh1and Rdidh1 (Table 13). This makes it possible to generate a Δidh1 PCRamplicon containing at its ends homologous recombination sequences ofthe isocitrate dehydrogenase IDH1 subunit gene locus.

The strains previously described, EQSC-002 (CEN.PK 1605 Δzwf1::hph) andCEN.PK 1605 (Mat a HISS leu2-3.112 trp1-289 ura3-52 MAL.28c) derivedfrom the commercial strain CEN.PK 113-7D (GenBank: JRIV00000000) aretransformed with the PCR fragment for inactivation of the IDH1 gene.

For the transformation reaction, strains EQSC-002 and CEN.PK1605 aregrown in a volume of 50 mL of complex rich medium YPD (yeast extractpeptone dextrose, here 20 g/L glucose) at 30° C. to an optical densityat 600 nm of 0.8. The cells are centrifuged for 5 minutes at 2,500 rpmat room temperature. The supernatant is removed and the cells areresuspended in 25 mL of sterile water and centrifuged again for 5minutes at 2,500 rpm at room temperature. After removing thesupernatant, the cells are resuspended in 400 μL of 100 mM sterilelithium acetate.

At the same time, a transformation mix is prepared in a 2 mL tube asfollows: 250 μL of 50% PEG, 10 μL of “carrier” DNA at 5 mg/mL, 36 μL of1 M lithium acetate, 10 μL of purified PCR reaction (deletion cassette)and 350 μL of water.

The resuspended cells (50 μL) are added to the transformation mixtureand incubated at 42° C. for 40 minutes in a water bath. Afterincubation, the tube is centrifuged for 1 minute at 5,000 rpm at roomtemperature and the supernatant is discarded. The cells are resuspendedin 2 mL of YPD (yeast extract peptone dextrose), transferred to a 14 mLtube and incubated for 2 hours at 30° C. at 200 rpm. The cells are thencentrifuged for 1 minute at 5,000 rpm at room temperature. Thesupernatant is removed and the cells are resuspended in 1 mL of sterilewater and centrifuged again for 1 minute and resuspended in 100 μL ofsterile water and spread on YPD+HygromycinB 200 μg/mL, 50nourseothricin.

The colonies obtained were genotyped for the validation of the deletionof the IDH1 gene and are called EQSC-003 (CEN.PK 1605 Δzwf1::hph,Δidh1::nat) and EQSC-005 (CEN.PK 1605 Δidh1::nat)

b) Inactivation of the PGK1 Gene in a Haploid Strain of Mating Type MATAlpha

The coding phase of the G418 resistance gene from the KanMX cassette(loxP-pAgTEF1-kanMX-tAgTEF1-loxP) contained on plasmid pUG6(P30114)—Euroscarf is amplified with the oligonucleotides Sdpgk1 andRdpgk1 (Table 13) to generate a Δpgk1 PCR amplicon containing at itsends homologous recombination sequences of the 3-phosphoglycerate kinasePGK1 gene locus.

Strain CEN.PK 1606 (Mat alpha HIS3 leu2-3.112 trp1-289 ura3-52 MAL.28c)derived from the commercial strain CEN.PK 113-7D (GenBank: JRIV00000000)is transformed with the PCR fragment for inactivation of the PGK1 gene.

For the transformation reaction, strain CEN.PK 1606 is grown in a volumeof 50 mL of complex rich medium YPD (yeast extract peptone dextrose,here 20 g/L glucose) at 30° C. to an optical density at 600 nm of 0.8.The cells are centrifuged for 5 minutes at 2,500 rpm at roomtemperature. The supernatant is removed and the cells are resuspended in25 mL of sterile water and centrifuged again for 5 minutes at 2,500 rpmat room temperature. After removing the supernatant, the cells areresuspended in 400 μL of 100 mM sterile lithium acetate.

At the same time, a transformation mix is prepared in a 2 mL tube asfollows: 250 μL of 50% PEG, 10 μL of “carrier” DNA at 5 mg/mL, 36 μL of1 M lithium acetate, 10 μL of purified PCR reaction (deletion cassette)and 350 μL of water.

The resuspended cells (50 μL) are added to the transformation mixtureand incubated at 42° C. for 40 minutes in a water bath. Afterincubation, the tube is centrifuged for 1 minute at 5,000 rpm at roomtemperature and the supernatant is discarded. The cells are resuspendedin 2 mL of YPGE (yeast extract peptone 20 g/L glycerol, 30 g/L ethanol),transferred to a 14 mL tube and incubated for 2 hours at 30° C. at 200rpm. The cells are then centrifuged for 1 minute at 5,000 rpm at roomtemperature. The supernatant is removed and the cells are resuspended in1 mL of sterile water and centrifuged again for 1 minute and resuspendedin 100 μL of sterile water and spread over YPGE+150 μg/mL G418.

The colonies obtained were genotyped for the validation of the deletionof the PGK1 gene and referenced EQSC-008 (CEN.PK 1605, Δpgk1::kan).

c) Construction of a Strain in which IDH1, ZWF1 and PGK1 have beenInactivated by Crossing

The haploid strains of opposite mating types EQSC-003 (CEN.PK 1605Δzwf1::hph, Δidh1::nat) and EQSC-008 (CEN.PK 1606 Δpgk1::kan) are grownovernight on agar medium:YPD (yeast extract peptone dextrose) for strainEQSC-008 and YPGE (yeast extract peptone glycerol ethanol) for strainEQSC003, at 30° C. Then the two strains are crossed by direct contact onYPGE (yeast extract peptone glycerol ethanol) agar medium+150 μg/mLG418+200 μg/mL hygromycin B. The G418 and hygromycin B double selectioneliminates the two parental strains, only the MAT a/MAT alpha,ZWF1/Δzwf1::hph, IDH1/Δidh1::nat, PGK1/Δpgk1::kan diploid strains growon this medium. An isolated diploid clone from this crossing iscollected. The presence of the three cassettes Δzwf1::hph, Δidh1::nat,Δpgk1::kan is validated by growth tests on YPGE (yeast extract peptoneglycerol ethanol) agar medium supplemented with 150 μg/mL G418 or 200μg/mL hygromycin B or 50 μg/mL nourseothricin. The strain obtained isreferenced EQSC-009 (CEN.PK 1607, MAT a/MAT alpha, ZWF1/Δzwf1::hph,IDH1/Δidh1::nat, PGK1/Δpgk1::kan).

The previously described strain EQSC-009 (CEN.PK 1607, MAT a/MAT alpha,ZWF1/Δzwf1::hph, IDH1/Δidh1::nat, PGK1/Δpgk1::kan) is grown on YPGE(yeast extract peptone glycerol ethanol) agar medium overnight at 30° C.The cells are then placed in liquid culture in a deficient medium(Sporulation Medium, 1% potassium acetate+leucine+uracil+tryptophan) toinduce meiosis of the diploid cells and thus lead to the formation oftetrads containing four haploid spores. The tetrads are spread on YNB.GEmedium (yeast nitrogen base, glycerol,ethanol)+leucine+uracil+tryptophan+1 g/L glutamic acid+20 mg/Lmethionine+40 mg/L cysteine and immediately dissected (using amicrodissector) to isolate the spores on the same medium. The spores aregerminated for several days at 30° C. The genetic content of the haploidcells thus obtained is tested by growth on selective media: YPGE (yeastextract peptone glycerol ethanol) supplemented with 150 μg/mL G418 or200 μg/mL hygromycin B or 50 μg/mL nourseothricin and their mating typeis tested by crossing with two tectrix strains of mating type MAT a orMAT alpha. The colonies obtained are genotyped for the validation of thedeletion of the PGK1, IDH1, ZWF1 genes and the absence of transcriptscorresponding to these genes is validated by real-time PCR after reversetranscription of ribonucleic acids. One of the strains obtained isreferenced EQSC-004 (CEN.PK 1606 MAT alpha Δzwf1::hph, Δidh1::nat,Δpgk1::kan)

d) Introduction of PRK-RuBisCO Enzymes

The six genes required for PRK-RuBisCO engineering (Table 15 below) arecloned on three plasmid vectors capable of autonomous replication, withcompatible origins of replication and each carrying a differentauxotrophic complementation gene, allowing the selection of strainscontaining the three plasmid constructs (see WO 2015107496). Two ofthese plasmids are single-copy with an ARS/CEN origin of replication andthe third is multicopy with a 2μ origin.

TABLE 15 Description of expression cassettes and plasmid compositionCodon Auxo- optimi- Termi- trophic GenBank zation Promoter nator orimarker Plasmids RbcL BAD78320.1 Yes TDH3p ADH1t 2μ URA3 pFPP45 RbcSBAD78319.1 Yes TEF1p PGK1t 2μ URA3 pFPP45 RbcX BAD80711.1 Yes TEF1pPGK1t ARS-CEN6 LEU2 pFPP56 GroES U00096 No PGI1p CYC1t ARS-CEN6 LEU2pFPP56 GroEL AP009048 No TDH3p ADH1t ARS-CEN6 LEU2 pFPP56 PRK BAD78757.1Yes Tet-OFF CYC1t ARS416-CEN4 TRP1 pFPP20 Empty Tet-OFF ARS416-CEN4 TRP1pCM185 Empty TEF1p PGK1t 2μ URA3 pV51TEF Empty ARS-CEN6 LEU2 pFL36

According to the transformation protocol previously described, strainEQSC-004 (CEN.PK 1606 Δzwf1::hph, Δidh1::nat, Δpgk1::kan) was grown in avolume of 50 mL of complex rich medium YPGE (yeast extract peptoneglycerol ethanol) at 30° C. to an optical density at 600 nm of 0.8. Thecells are centrifuged for 5 minutes at 2,500 rpm at room temperature.The supernatant is removed and the cells are resuspended in 25 mL ofsterile water and centrifuged again for 5 minutes at 2,500 rpm at roomtemperature. After removing the supernatant, the cells are resuspendedin 400 μL of 100 mM sterile lithium acetate.

At the same time, a transformation mix is prepared in a 2 mL tube asfollows: 250 μL of 50% PEG, 10 μL of “carrier” DNA at 5 mg/mL, 36 μL of1 M lithium acetate, 10 μL (3 μg) of a combination ofpFPP45+pFPP56+pFPP20 and 350 μL of water.

The resuspended cells (50 μL) are added to the transformation mixtureand incubated at 42° C. for 40 minutes in a water bath. Afterincubation, the tube is centrifuged for 1 minute at 5,000 rpm at roomtemperature and the supernatant is discarded. The cells are resuspendedin 2 mL of YPGE (yeast extract peptone glycerol ethanol)+2 mg/Ldoxycycline, transferred into a 14 mL tube and incubated for 2 hours at30° C. at 200 rpm. The cells are then centrifuged for 1 minute at 5,000rpm at room temperature. The supernatant is removed and the cells areresuspended in 1 mL of sterile water and centrifuged again for 1 minuteand resuspended in 100 μL of sterile water and spread over YNB.GE (yeastnitrogen base, glycerol, ethanol)+1 g/L glutamic acid+20 mg/Lmethionine+40 mg/L cysteine+2 mg/L doxycycline. The strain obtained isreferenced: EQSC-006 (CEN.PK 1606 Δzwf1::hph, Δidh1::nat, Δpgk1::kan)(pFPP45+pFPP56+pFPP20).

According to the transformation protocol previously described, strainEQSC-005 (CEN.PK 1605 Δidh1::nat) was grown in a volume of 50 mL ofcomplex rich medium YPGE (yeast extract peptone glycerol ethanol) at 30°C. to an optical density at 600 nm of 0.8. The cells are centrifuged for5 minutes at 2,500 rpm at room temperature. The supernatant is removedand the cells are resuspended in 25 mL of sterile water and centrifugedagain for 5 minutes at 2,500 rpm at room temperature. After removing thesupernatant, the cells are resuspended in 400 μL of 100 mM sterilelithium acetate.

At the same time, a transformation mix is prepared in a 2 mL tube asfollows: 250 μL of 50% PEG, 10 μL of “carrier” DNA at 5 mg/mL, 36 μL of1 M lithium acetate, 10 μL (3 μg) of a combination ofpV51TEF+pFL36+pCM185 and 350 μL of water.

The resuspended cells (50 μL) are added to the transformation mixtureand incubated at 42° C. for 40 minutes in a water bath. Afterincubation, the tube is centrifuged for 1 minute at 5,000 rpm at roomtemperature and the supernatant is discarded. The cells are resuspendedin 2 mL of YPD (yeast extract peptone dextrose), transferred to a 14 mLtube and incubated for 2 hours at 30° C. at 200 rpm. The cells are thencentrifuged for 1 minute at 5,000 rpm at room temperature. Thesupernatant is removed and the cells are resuspended in 1 mL of sterilewater and centrifuged again for 1 minute and resuspended in 100 μL ofsterile water and spread on YNBD (yeast nitrogen base dextrose)+2 mg/Ldoxycycline. The strain obtained is referenced: EQSC-007 (CEN.PK 1605Δidh1::nat) (pV51TEF+pFL36+pCM185).

d) Adaptation and Evolution Phase of Strains EQSC-006 and EQSC-007

Adaptation of strains EQSC-006 and EQSC-007 to growth on YNB (yeastnitrogen base) liquid medium with glucose and CO₂.

Batch-mode cultures in Erlenmeyer flasks are carried out with theappropriate culture medium and a 10% exogenous CO₂ supply, in a shakingincubator (120 rpm, 30° C.), with inoculation at 0.05 OD 600 nm measuredusing an EON spectrophotometer (BioTek Instruments). The strain ofinterest is grown on YNB+CSM-LUW medium with 10 g/L glycerol and 7.5 g/Lethanol, +50 mg/L glutamate under conditions where PRK expression is notinduced.

After obtaining a sufficient quantity of biomass, cultures with a volumegreater than or equal to 50 mL in Erlenmeyer flasks of at least 250 mLare inoculated in order to adapt the strain to the use of thePRK/RuBisCO engineering. This adaptation is carried out on YNB+CSM-LUWculture medium with 20 g/L glucose, 50 mg/L glutamate and an exogenousCO₂ supply as described above.

After observation of a significant growth start, the strains are adaptedto a minimum mineral medium free of all amino acids except thoseindicated below, and nitrogenous bases included in the CSM-LUW, i.e.only YNB with, in final concentrations, 20 g/L glucose, 1 g/L glutamate,40 mg/L L-cysteine and 20 mg/L L-methionine and an exogenous CO₂ supplyas described above.

e) Production of Citrate in Erlenmeyer Flasks

Saccharomyces cerevisiae strain EQSC-006, with a deletion in theglycolytic pathway at the PGK1 gene, in the oxidative part of thepentose phosphate pathway and in the Krebs cycle, is grown to producecitrate without CO₂ loss, using PRK and RuBisCO. This strain of interestis compared with a reference strain EQSC-007 producing citrate followinginactivation of the IDH1 gene, without deletion of PGK1 or ZWF1 oraddition of PRK and RuBisCO.

Strains EQSC-006 (CEN.PK 1605 Δzwf1::hph, Δidh1::nat, Δpgk1::kan,pFPP45+pFPP56+pFPP20) and EQSC-007 (CEN.PK 1605 Δidh1::nat,pV51TEF+pFL36+pCM185) were cultured in yeast nitrogen base (YNB) mediumsupplemented with 20 g/L D-glucose (YNB D20).

In order to establish the citrate to glucose mass yields, a pre-culturecontaining 20 mL of culture medium was inoculated at 0.05 OD_(600 nm)into a 250 mL baffled Erlenmeyer flask, shaken at 120 rpm at 30° C. Fromthe first pre-culture, 50 mL of medium was inoculated at 0.05OD_(600 nm) into a 250 mL Erlenmeyer flask and shaken at 120 rpm, at 30°C. The culture was carried out in Erlenmeyer flasks (500 mL, baffled)from the second pre-culture, inoculated at 0.05 OD_(600 nm) into 100 mLof the same medium, at 30° C., 120 rpm. Growth was monitored bymeasuring turbidity at 600 nm.

For citrate quantification, 500 μL of culture medium was collected,centrifuged at 5,000 g, 5 min, 4° C. The supernatant was filtered(Minicart RC4, Sartorius 0.45 μm) and stored in a flask at −20° C.before HPLC analysis (Thermo Scientific UltiMate 3000 HPLC) coupled to asingle quadrupole mass spectrometer. Each sample (20 μL) was injectedinto an Aminex HPX-87H H⁺ column, 300 mm×7.8 mm (Bio-Rad, 125-0140). Anisocratic elution at a flow rate of 0.5 mL/min was carried out with anaqueous solution of 0.037% formic acid (v/v) whose pH was adjusted to4.5 with ammonium hydroxide. The column oven temperature was 65° C. Themass spectrometry analytical conditions were: negative electrospraymode, source temperature 450° C., needle voltage 3 kV, cone voltage 50V. A seven-point external calibration was performed using a commercialsodium citrate solution.

To quantify the glucose consumed by the strains, 500 μL of the culturemedium was collected, at the same culture OD_(600 nm) as for citratequantification, centrifuged at 5,000 g, 5 min at 4° C. The supernatantwas filtered (Minicart RC4, Sartorius 0.45 μm) and stored in a flask at−20° C. The glucose contained in this sample was quantified by HPLC-RIUltiMate 3000 (Thermo Scientific) equipped with a pump, an 8° C.refrigerated autosampler and a refractive index (RI) detector (PrecisionInstruments IOTA 2). A Rezex ROA-Organic Acid H⁺ column (8%) 150×7.8 mm,8 μm particle size (Phenomenex, 00H-0138-KO) was used with a Carbo-H4×3.0 mm pre-column. The column oven temperature was 35° C. and the flowrate was set at 0.5 mL/min A 30 min isocratic elution was performed withan aqueous mobile phase at 5 mM H₂SO₄. A volume of 20 μL was injectedfor each sample. The identification of the compounds was based on thecomparison of retention times with standards. The external calibrationincluded 10 points of variable glucose concentration (0 to 20 g/L).

The Y_(citrate/Glc) mass yield was calculated in grams of citrateproduced per gram of glucose consumed for both strains EQSC-006 andEQSC-007,

$Y_{citrat{e/G}lc} = {\frac{{citrate}\mspace{14mu} \left( {{mg}\text{/}{L{aqueous}}} \right)}{{glucose}\mspace{14mu} \left( {{mg}\text{/}{Laqueous}} \right)}.}$

TABLE 16 Mass yield, citrate to D-glucose Y_(citrate/glucose) YieldStrains (×10⁻³) (g/g) improvement EQSC-006 2.1 +19.5% EQSC-007 1.8

A 19.5% increase in the citrate to D-glucose mass yield was observed forstrain EQSC-006 compared with control strain EQSC-007.

Example 4: Improvement of Glutamate Production in E. coli

Deletion of the alpha-ketoglutarate dehydrogenase gene increasesglutamate production (Usuda et al. J Biotechnol. 2010 May 3;147(1):17-30. doi: 10.1016/j.jbiotec.2010.02.018).

In these examples, Escherichia coli strain K12 MG1655 with a deletedsucA gene was used. This strain is derived from a gene deletion bank(Baba et al. Mol Syst Biol. 2006; 2:2006.0008) in Escherichia coli andsupplied by the coli Genetic Stock Center under the name JW0715-2 andwith reference 8786. (JW0715-2: MG1655 ΔsucA::Kan)

4A] Improvement of Glutamate Production by Inactivation of Glycolysis

a) Removal of the Selection Cassette by Specific Recombination of FTRRegions by Flp Recombinase

In order to be able to reuse the same deletion strategy as that used toconstruct strain JW0715-2 above (Rodriguez et al., 2016), the selectioncassette was deleted using a recombinase.

Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit by Gene Bridges) is transformed byelectroporation according to the kit protocol. The cells are selected onLB agar supplemented with 0.2% glucose, 0.0003% tetracycline and addedwith 0.3% L-arabinose. A counter-selection of the clones obtained iscarried out by verifying that they are no longer able to grow on thesame medium supplemented with 0.0015% kanamycin.

The strain obtained is called EQ.EC002: MG1655 ΔsucA

b) Deletion of the Edd-Eda Operon Encoding the Entner-DoudoroffMetabolic Pathway

The deletion of the edd-eda operon is performed by homologousrecombination and the use of the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit (Gene Bridges) according to the supplier'sprotocol.

-   1. Oligonucleotides designed to amplify an FRT-PKG-gb2-neo-FRT    resistance gene expression cassette and having a 5′ sequence    homologous over 50 nucleotides to the adjacent regions of the    deletion locus, i.e. at positions 1932065-1932115 and    1934604-1934654 on the chromosome thus generating recombination arms    of the cassette on the bacterial genome on either side of the entire    operon.-   2. The Escherichia coli K-12 strain EQ.EC002 is transformed by    electroporation with plasmid pRedET according to the kit protocol.    The colonies obtained are selected on rich complex medium LB agar    with 0.2% glucose, 0.0003% tetracycline.-   3. Transformation of the amplicon obtained in the first step in the    presence of RedET recombinase, induced by 0.3% arabinose in liquid    LB for 1 h. To that end, a second electroporation of the cells    expressing RedET by the deletion cassette is performed and the    colonies are selected on LB agar supplemented with 0.2% glucose,    0.0003% tetracycline and added with 0.3% L-arabinose and 0.0015%    kanamycin.-   4. Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene    Deletion Red®/ET® Recombination Kit by Gene Bridges) is transformed    by electroporation according to the kit protocol. The cells are    selected on LB agar supplemented with 0.2% glucose, 0.0003%    tetracycline and added with 0.3% L-arabinose. A counter-selection of    the clones obtained is carried out by verifying that they are no    longer able to grow on the same medium supplemented with 0.0015%    kanamycin.-   5. The strain obtained is called EQ.EC003: MG1655 ΔsucA Δedd-eda

c) Deletion of the gapA Gene

The deletion of the gapA gene is performed by homologous recombinationand the use of the Quick & Easy E. coli Gene Deletion Red®/ET®Recombination Kit (Gene Bridges) according to the supplier's protocol.

-   1. Oligonucleotides designed to amplify an FRT-PKG-gb2-neo-FRT    resistance gene expression cassette and having a 5′ sequence    homologous over 50 nucleotides to the adjacent regions of the    deletion locus, i.e. the coding phase of the gene (gapA) (GenBank:    X02662.1) thus generating recombination arms of the cassette on the    bacterial genome.-   2. The Escherichia coli K-12 strain EQ.EC003 is transformed by    electroporation with plasmid pRedET according to the kit protocol.    The colonies obtained are selected on rich complex medium LB agar    with 0.2% glucose, 0.0003% tetracycline.-   3. Transformation of the amplicon obtained in the first step in the    presence of RedET recombinase which will be induced by 0.3%    arabinose in liquid LB for 1 h. To that end, a second    electroporation of the cells expressing RedET by the deletion    cassette is performed and the colonies are selected on LB agar    supplemented with 0.2% glycerol and 0.3% pyruvate, 0.0003%    tetracycline and added with 0.3% L-arabinose and 0.0015% kanamycin.

Deletions are verified by genotyping and sequencing and the name of thestrains obtained is

-   -   EQ.EC002: MG1655 ΔsucA    -   EQ.EC003: MG1655 ΔsucA Δedd-eda    -   EQ.EC004: MG1655 ΔsucA Δedd-eda ΔgapA::kan

d) Insertion of the Engineering Required for CO₂ Fixation

For the recombinant expression of the different components of a type IRuBisCO in E. coli, the genes described in Table 17 below are cloned asa synthetic operon containing the genes described in Table 18 below.

TABLE 17 Genes encoding a PRK and type I RuBisCO system Genes GenBankOrganism rbcL BAD78320.1 Synechococcus elongatus rbcS BAD78319.1Synechococcus elongatus rbcX BAD80711.1 Synechococcus elongatus PrkBAD78757.1 Synechococcus elongatus

TABLE 18 Composition of the expression cassettes Structure of thesynthetic operon in vector pZA11 Plasmid Gene A RBS1 Gene B RBS2 Gene CRBS3 Gene D RBS4 Gene E pZA11 pEQEC005 rbcS D rbcL B rbcX F pEQEC006rbcS D rbcL B rbcX F prk pEQEC008 prk

To control the expression level of these genes, ribosome bindingsequences (RBS) presented in Table 19 below, with variable translationefficiencies (Levin-Karp et al., ACS Synth Biol. 2013 Jun. 21;2(6):327-36. doi: 10.1021/sb400002n; Zelcbuch et al., Nucleic Acids Res.2013 May; 41(9):e98) are inserted between the coding phase for eachgene. The succession of each coding phase interspersed by an RBSsequence is constructed by successive insertions into a pZA11 vector(Expressys) that contains a PLtetO-1 promoter, a p15A origin ofreplication and an ampicillin resistance gene.

TABLE 19 RBS intercistronic sequences Name RBS sequencesA (SEQ ID NO: 9) AGGAGGTTTGGA B (SEQ ID NO: 10) AACAAAATGAGGAGGTACTGAGC (SEQ ID NO: 11) AAGTTAAGAGGCAAGA D (SEQ ID NO: 12) TTCGCAGGGGGAAGE (SEQ ID NO: 13) TAAGCAGGACCGGCGGCG F (SEQ ID NO: 14) CACCATACACTG

Several strains are produced by electroporating the different vectorspresented according to the above plan

EQ.EC 005→(EQ.EC 003+pZA11): MG1655 ΔsucA Δedd-eda

EQ.EC 006→(EQ.EC 004+pEQEC005): MG1655 ΔsucA Δedd-eda ΔgapA::kan(RuBisCO)

EQ.EC 007→(EQ.EC 004+pEQEC006): MG1655 ΔsucA Δedd-eda ΔgapA::kan(RuBisCO+PRK)

EQ.EC 009→(EQ.EC 004+pEQEC008): MG1655 ΔsucA Δedd-eda ΔgapA::kan (PRK)

Clones are selected on LB medium supplemented with 2 g/L glycerol and 5g/L pyruvate and with 100 mg/L ampicillin. After obtaining a sufficientquantity of biomass, cultures with a volume greater than or equal to 50mL in a minimum 250 mL Erlenmeyer flask are inoculated in order to adaptthe strain to the use of the PRK/RuBisCO engineering. This adaptation iscarried out on LB culture medium with 2 g/L glucose, and an exogenousCO₂ supply at 37° C. as described above.

e) Glutamate Production

For glutamate production, cells from 500 mL of LB culture are inoculatedinto 20 mL of MS medium (40 g/L glucose, 1 g/L MgSO₄.7H₂O, 20 g/L(NH₄)₂SO₄, 1 g/L KH₂PO₄, 10 mg/L FeSO₄.7H₂O, 10 mg/L MnSO₄.7H₂O, 2 g/Lyeast extract, 30 g/L CaCO₃, 100 mg/L ampicillin at a pressure of 0.1atmosphere CO₂.

Residual glutamate and glucose are measured with a bioanalyzer (SakuraSeiki). The carbon yield Y_(p/s) is calculated in grams of glutamateproduced per gram of glucose consumed.

This yield increases significantly by 10% for strains EQ.EC 007(RuBisCO+PRK) compared with the control strains EQ.EC 005 (empty), EQ.EC006 (RuBisCO only). The control strain EQ.EC 009 (PRK alone) is notviable.

4B] Improvement of Production by Inactivation of Glycolysis and of thePentose Phosphate Oxidative Pathway

a) Removal of the Selection Cassette by Specific Recombination of FTRRegions by Flp Recombinase

This step is performed in the same way as example 4A] above.

The strain obtained is called EQ.EC002: MG1655 ΔsucA

b) Deletion of the zwf Gene

The deletion of the zwf gene (GeneID: 946370) is performed by homologousrecombination and the use of the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit (Gene Bridges) according to the supplier'sprotocol, as detailed in Example 4A].

The strain obtained is called EQ.EC010: MG1655 ΔsucA Δzwf

c) Deletion of the gapA Gene

The deletion of the gapA gene in the Escherichia coli K-12 strainEQ.EC010 is performed by homologous recombination and the use of theQuick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (GeneBridges) according to the supplier's protocol, as detailed in Example4A].

Deletions are verified by genotyping and sequencing and the name of thestrains obtained is:

-   -   EQ.EC002: MG1655 ΔsucA    -   EQ.EC010: MG1655 ΔsucA Δzwf    -   EQ.EC011: MG1655 ΔsucA Δzwf ΔgapA

d) Insertion of the Engineering Required for CO₂ Fixation

For the recombinant expression of the different components of thefunctional PRK/RuBisCO system in E. coli, the genes described in Table20 and encoding a type I RuBisCO, a phosphoribulokinase, a chaperone anda carbonic anhydrase are cloned as a synthetic operon containing thegenes described above (Table 21).

TABLE 20 Genes encoding a type I RuBisCO, a phosphoribulokinase and acarbonic anhydrase Genes GenBank Organism rbcL BAD78320.1 Synechococcuselongatus rbcS BAD78319.1 Synechococcus elongatus rbcX BAD80711.1Synechococcus elongatus Prk BAD78757.1 Synechococcus elongatus icfAWP_011378036.1 Synechococcus elongatus

TABLE 21 Plasmid names and expression cassette composition Structure ofthe synthetic operon in vector pZA11 Plasmid Gene A RBS1 Gene B RBS2Gene C RBS3 Gene D RBS4 Gene E pZA11 pEQEC006 rbcS D rbcL B rbcX F prkpEQEC007 rbcS D rbcL B rbcX F prk A icfA

To control the expression level of these genes, ribosome bindingsequences (RBS) presented in Table 17 (see Example 4A]), with variabletranslation efficiencies (Levin-Karp et al., ACS Synth Biol. 2013 Jun.21; 2(6):327-36. doi: 10.1021/sb400002n; Zelcbuch et al., Nucleic AcidsRes. 2013 May; 41(9):e98) are inserted between the coding phase of eachgene. The succession of each coding phase interspersed by an RBSsequence is constructed by successive insertions into a pZA11 vector(Expressys) that contains a PLtetO-1 promoter, a p15A origin ofreplication and an ampicillin resistance gene. The addition of acarbonic anhydrase (icfA) also allows an inter-conversion of bicarbonateions into available CO₂ molecules and improves the efficiency ofRuBisCO.

Several strains are produced by electroporating the different vectorspresented according to the plan below

EQ.EC 012→(EQ.EC 002+pZA11): MG1655 ΔsucA

EQ.EC 014→(EQ.EC 011+pEQEC006): MG1655 ΔsucA Δzwf ΔgapA (RuBisCO+PRK)

EQ.EC 015→(EQ.EC 011+pEQEC007): MG1655 ΔsucA Δzwf ΔgapA(RuBisCO+PRK+carbonic anhydrase)

After transformation, clones are selected on LB glycerol, pyruvatemedium supplemented with 100 mg/L ampicillin. An adaptation andevolution phase of the strains with PRK and RuBisCO engineering isperformed as described in Example 4A].

e) Glutamate Production

For glutamate production, cells from 500 mL of LB culture are inoculatedinto 20 mL of MS medium (40 g/L glucose, 1 g/L MgSO₄.7H₂O, 20 g/L(NH4)₂SO₄, 1 g/L KH₂PO₄, 10 mg/L FeSO₄.7H₂O, 10 mg/L MnSO₄.7H₂O, 2 g/Lyeast extract, 30 g/L CaCO₃, 100 mg/L ampicillin at a pressure of 0.1atmosphere CO₂.

Residual glutamate and glucose are measured with a bioanalyzer (YSIInc.). The carbon yield Y_(p/s) is calculated in grams of glutamateproduced per gram of glucose consumed.

This yield increases significantly by 15% for strains EQ.EC 014(RuBisCO+PRK) and EQ.EC 015 (RuBisCO+PRK+carbonic anhydrase) comparedwith the control strains EQ.EC 012 (empty).

4C] Improvement of Production by Inactivation of Glycolysis andOxidative Pentose Phosphate Pathway, and Overexpression of PyruvateDecarboxylase and Glutamate Dehydrogenase.

a) Removal of the Selection Cassette by Specific Recombination of FTRRegions by Flp Recombinase

This step is performed in the same way as example 4A] above.

The strain obtained is called EQ.EC002: MG1655 ΔsucA

b) Deletion of the zwf Gene

The deletion of the zwf gene (GeneID: 946370) is performed by homologousrecombination and the use of the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit (Gene Bridges) according to the supplier'sprotocol, as detailed in Example 4A]. The strain obtained is calledEQ.EC010: MG1655 ΔsucA Δzwf

c) Deletion of the gapA Gene

The deletion of the gapA gene in the Escherichia coli K-12 strainEQ.EC010 is performed by homologous recombination and the use of theQuick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (GeneBridges) according to the supplier's protocol, as detailed in Example4A]. Deletions are verified by genotyping and sequencing and the name ofthe strains obtained is:

-   -   EQ.EC002: MG1655 ΔsucA    -   EQ.EC010: MG1655 ΔsucA Δzwf    -   EQ.EC011: MG1655 ΔsucA Δzwf ΔgapA

d) Insertion of the Engineering Necessary for CO₂ Fixation

For the recombinant expression of the different components of thefunctional PRK/RuBisCO system in E. coli, the genes described in Table22 and encoding a type II RuBisCO, a phosphoribulokinase and a carbonicanhydrase are cloned as a synthetic operon containing the genesdescribed above (Table 23).

TABLE 22 Genes encoding a type II RuBisCO, a phosphoribulokinase, acarbonic anhydrase, a glutamate dehydrogenase and a pyruvate carboxylaseGenes GenBank Organism cbbM YP_427487.1 Rhodospirillum rubrum PrkBAD78757.1 Synechococcus elongatus CA YP_427143.1 Rhodospirillum rubrumgdhA NP_416275.1 Escherichia coli K-12 pycA NP_389369.1 Bacillussubtilis

TABLE 23 Plasmid names and expression cassette composition Structure ofthe synthetic operon in vector pZA11 Plasmid Gene A RBS1 Gene B RBS2Gene C RBS3 Gene D RBS4 Gene E pZA11 pEQEC009 cbbM B gdhA C pycA E prkpEQEC010 cbbM B gdhA C pycA E prk D CA pEQEC011 B gdhA C pycA

To control the expression level of these genes, ribosome bindingsequences (RBS) presented in Table 17 (see Example 4A]), with variabletranslation efficiencies, are inserted between the coding phase of eachgene. The succession of each coding phase interspersed by an RBSsequence is constructed by successive insertions into a pZA11 vector(Expressys) that contains a PLtetO-1 promoter, a p15A origin ofreplication and an ampicillin resistance gene. The addition of aglutamate dehydrogenase (gdhA) and a pyruvate carboxylase (pycA) allowsa better production of glutamic acid. The addition of a carbonicanhydrase (CA) also allows an interconversion of bicarbonate ions intoavailable CO₂ molecules and improves the efficiency of RuBisCO.

Several strains are produced by electroporating the different vectorspresented according to the plan below:

EQ.EC 016→(EQ.EC 002+pEQEC011): MG1655 ΔsucA (glutamatedehydrogenase+pyruvate carboxylase)

EQ.EC 017→(EQ.EC 011+pEQEC009): MG1655 ΔsucA Δzwf ΔgapA(RuBisCO+PRK+glutamate dehydrogenase+pyruvate carboxylase)

EQ.EC 018→(EQ.EC 011+pEQEC010): MG1655 ΔsucA Δzwf ΔgapA(RuBisCO+PRK+carbonic anhydrase+glutamate dehydrogenase+pyruvatecarboxylase+carbonic anhydrase)

After transformation, clones are selected on LB glycerol, pyruvatemedium supplemented with 100 mg/L ampicillin. An adaptation andevolution phase of the strains with PRK and RuBisCO engineering isperformed as described in Example 4A].

e) Glutamate Production

For glutamate production, cells from 500 mL of LB culture are inoculatedinto 20 mL of MS medium (40 g/L glucose, 1 g/L MgSO₄.7H₂O, 20 g/L(NH₄)₂SO₄, 1 g/L KH₂PO₄, 10 mg/L FeSO₄.7H₂O, 10 mg/L MnSO₄.7H₂O, 2 g/Lyeast extract, 30 g/L CaCO₃, 100 mg/L ampicillin at a pressure of 0.1atmosphere CO₂.

Residual glutamate and glucose are measured with a bioanalyzer (YSIInc.). The carbon yield Y_(p/s) is calculated in grams of glutamateproduced per gram of glucose consumed.

This yield increases significantly by 15% for strains EQ.EC 017 andEQ.EC 018 compared with the control strain EQ.EC 016.

Example 5: Improvement of Polyhydroxybutyrate Production in C. necator

The increase in reducing power obtained through the geneticmodifications proposed according to the invention may also have aconsiderable gain over existing metabolic pathways.

This is the case for the bacterial strain Cupriavidus necator ATCC 17699which naturally produces polyhydroxybutyrate (PHB). This bacterium iscapable of developing under both autotrophic and heterotrophicconditions. The deletion of the gapA gene (glyceraldehyde-3-phosphatedehydrogenase NC_008313.1) diverts the metabolic flux to the pentosephosphate pathway and increases the pool of NADPH reduced nucleotidesthus increasing the PHB production yield.

This C. necator H16 strain has a megaplasmid pHG1 and two chromosomes.The deletion of the gapA gene is performed by generating a vectorcontaining the Bacillus subtilis suicide gene sacB for Gram-negativebacteria (Quandt et al., Gene. 1993 May 15; 127(1):15-21; Lindenkamp etal., Appl Environ Microbiol. 2010 August; 76(16):5373-82 and ApplEnviron Microbiol. 2012 August; 78(15):5375-83).

a) Inactivation of the Entner-Doudoroff Metabolic Pathway

Two PCR amplicons corresponding to adjacent regions of the edd and edagenes (upstream of edd and downstream of eda) are cloned by restrictionaccording to the procedure described in Srinivasan et al. (Appl EnvironMicrobiol. 2002 December; 68(12):5925-32), in plasmid pJQ200mp18Cm.

The modified plasmid pJQ200mp18Cm::Δedd-eda is then transformed into anE. coli strain S17-1 by the calcium chloride transformation method. Thetransfer of genetic material into C. necator is done by conjugation bydepositing on agar a spot of C. necator culture on a dish containing acell monolayer of S17-1 bacteria. Selection is made on nutrient broth(NT) medium at 30° C. in the presence of 10% sucrose for purposes ofselection (Hogrefe et al., J Bacteriol. 1984 April; 158(1):43-8) andvalidated on a mineral medium containing 50 μg/mL chloramphenicol.

The deletions are validated by genotyping and sequencing. The resultingstrain EQCN_002 therefore has deletions of the genes of theEntner-Doudoroff metabolic pathway edd-eda. EQCN_002: H16 Δedd-eda.

b) Inactivation of the Glycolysis Pathway

Two PCR amplicons corresponding to adjacent regions of the gapA gene arecloned by restriction according to the procedure described in Lindenkampet al. 2012, in plasmid pjQ200mp18Tc.

The modified plasmid pjQ200mp18Tc::ΔgapA is then transformed into an E.coli strain S17-1 by the calcium chloride transformation method. Thetransfer of genetic material is done by conjugation by depositing onagar a spot of C. necator culture on a plate containing a cell monolayerof S17-1 bacteria. Selection is made on nutrient broth (NT) medium at30° in the presence of 10% sucrose for purposes of selection (Hogrefe etal., J Bacteriol. 1984 April; 158(1):43-8) and validated on a mineralmedium containing 25 μg/mL tetracycline.

The deletions are validated by genotyping and sequencing. The strainobtained, EQCN_003, therefore has a deletion of the gapA gene. EQCN_003:H16 Δedd-eda ΔgapA.

Strain EQCN_003, with a deletion in the glycolytic pathway at the gapAgene and in the Entner-Doudoroff pathway at the edd-eda genes, is grownto improve PHB production yield by fixing exogenous CO₂ via the use ofthe PRK and RuBisCO enzymes.

b) Production of PHB in a Bioreactor

The inoculum from a frozen stock is spread on solid medium at a rate of50 to 100 μL from a cryotube incubated at 30° C. for 48 to 96 h in thepresence of fructose. The expression of genes encoding RuBisCO and PRKare maintained in C. necator under heterotrophic aerobic conditions (RieShimizu et al., Sci Rep. 2015; 5: 11617. Published online 2015 Jul. 1).

Batch cultures in Erlenmeyer flasks (10 mL in 50 mL, then 50 mL in 250mL) are carried out with the appropriate culture medium, in 20 g/Lfructose and a 10% exogenous CO₂ supply in a shaking incubator (100-200rpm, 30° C.), with a minimum inoculation of 0.01.

The strain of interest EQCN_003 improving PHB production yield iscompared with a reference strain H16 naturally accumulating PHB underheterotrophic conditions in the presence of a nutritional limitation.

The productivity of the strains is compared in bioreactors. Culturescarried out in bioreactors are seeded from solid and/or liquidamplification chains in Erlenmeyer flasks under the conditions describedabove. The bioreactors, of type My-control (Applikon Biotechnology,Delft, Netherlands) 750 mL or Biostat B (Sartorius Stedim, Göttingen,Germany) 2.5 L, are seeded at a density equivalent to 0.01 OD_(620 nm).

The accumulation of PHB is decoupled from growth. The culture isregulated at 30° C., aeration is between 0.1 VVM (gas volume/liquidvolume/min) and 1 VVM in order to maintain a minimum dissolved oxygenconcentration above 20% (30° C., 1 bar), shaking is adapted according tothe scale of the bioreactor used. The inlet gas flow consists of airoptionally supplemented with CO₂. CO₂ supplementation is between 1% and10%. The pH is adjusted to 7 with a 14% or 7% ammonia solution. Thefed-batch culture method allows a supply of non-limiting carbonsubstrate combined with a limitation of phosphorus or nitrogen, whilemaintaining a constant carbon/phosphorus or carbon/nitrogen ratio. PHBextraction and quantification are performed according to the method ofBrandl et al. (Appl Environ Microbiol. 2013 July; 79(14):4433-9). Theprotocol consists in adding 1 mL of chloroform to 10 mg of lyophilizedcells, followed by 850 μL of methanol and 150 μL of sulfuric acid. Themixture is heated for 2.5 h at 100° C., cooled and 500 μL of water isadded. The two phases are separated by centrifugation and the organicphase is dried by adding sodium sulfate The samples are filtered andanalyzed as described by Müller et al. (Appl Environ Microbiol. 2013July; 79(14):4433-9).

A comparison of wild-type C. necator H16 cultures and strain EQCN_003:H16 Δedd-eda ΔgapA shows a 5% increase in carbon yield, correspondinghere to the ratio grams of PHB per gram of fructose consumed.

Example 6: Improvement of GABA Production in E. coli

An Escherichia coli K-12 strain, genetically modified to increase theyield of its glutamate production according to example 4B], can also bemodified to allow the constitutive expression of a glutamatedecarboxylase gadB (Gene ID: 946058) and thus increase the productionyield of γ-aminobutyric acid.

The deletion of the alpha-ketoglutarate dehydrogenase gene alsoincreases glutamate production (Usuda et al. J Biotechnol. 2010 May 3;147(1):17-30. doi: 10.1016/j.jbiotec.2010.02.018).

In this example, the following strains are used, obtained from example4B]:

-   -   EQ.EC002: MG1655_ΔsucA    -   EQ.EC010: MG1655_ΔsucA Δzwf    -   EQ.EC011: MG1655 ΔsucA Δzwf ΔgapA

a) Constitutive Overexpression of the gadB Gene

Overexpression of the gadB gene is subcloned into a bacterial expressionvector pZE21MCS (EXPRESSYS). This vector has a ColE1 origin ofreplication and a kanamycin antibiotic resistance gene.

Rapidly, the coding phase of the gadB gene (Gene ID: 946058) isamplified from the genome of strain MG1655 ΔsucA with primers homologousto the Escherichia coli K-12 genome covering positions 1570595 to1570645 and 1572095 to 1572045. Each of these primers is coupled tofloating sequences homologous over 18 nucleotides at the ends of thefragment obtained by amplifying vector pZE21MCS excluding the multiplecloning site. The two amplicons are combined according to the protocolof the In-Fusion® HD Cloning Kit User Manual—Clontech to form plasmidpEQEC030 allowing the constitutive overexpression of the gadB gene.

b) Insertion of the Engineering Required for CO₂ Fixation

For the recombinant expression of the different components of afunctional type I RuBisCO in E. coli, the genes described in Table 17(Example 4A]), are cloned as a synthetic operon following theconstruction structure described in Table 22.

Assembly of the Different Vectors

The coding sequences (CDS) of the genes described in Table 24 areamplified and assembled into blocks according to the protocol providedwith the NEBuilder® HiFi DNA Assembly Master Mix Kit (E2321) so as toobtain three integration blocks described in Table 24. Each block isthen amplified according to the protocol of the In-Fusion® HD CloningKit User Manual—Clontech to form the plasmids described below in Table24.

TABLE 24 Composition of expression cassettes Structure of the syntheticoperon in vector pZA11 Block I Block II Block III Plasmid CDS A RBS1 CDSB RBS2 CDS C RBS3 CDS D pZA11 pEQEC006 rbcS D rbcL B rbcX F prk

To control the expression level of these genes, ribosome bindingsequences (RBS) presented in Table 19 (Example 4B]), with variabletranslation efficiencies (Levin-Karp et al., ACS Synth Biol. 2013 Jun.21; 2(6):327-36. doi: 10.1021/sb400002n; Zelcbuch et al., Nucleic AcidsRes. 2013 May; 41(9):e98) are inserted between the coding phase for eachgene. The succession of each coding phase interspersed by an RBSsequence is constructed by successive insertions into a pZA11 vector(Expressys) that contains a PLtetO-1 promoter, a p15A origin ofreplication and an ampicillin resistance gene. The addition of aglutamate decarboxylase (gadB) also allows a conversion of glutamate togamma-aminobutyrate (GABA).

Several strains are produced by electroporating the different vectorspresented according to the plan below

-   EQ.EC 013→(EQ.EC 002+pZA11+pEQ030): MG1655 ΔsucA+(gadB)-   EQ.EC 020→(EQ.EC 011+pEQ030+pEQEC006): MG1655 ΔsucA Δzwf    ΔgapA+(gadB)+(RuBisCO+PRK)

After transformation, clones are selected on LB glycerol, pyruvatemedium supplemented with 100 mg/L ampicillin and 30 mg/L kanamycin. Anadaptation and evolution phase of the strains with PRK and RuBisCOengineering is performed as described in Example 4A].

c) GABA Production

For the production of GABA, cells from 500 mL of LB culture areinoculated into 20 mL of MS medium (40 g/L glucose, 1 g/L MgSO₄.7H₂O, 20g/L (NH₄)₂SO₄, 1 g/L KH₂PO₄, 10 mg/L FeSO₄.7H₂O, 10 mg/L MnSO₄.7H₂O, 2g/L yeast extract, 30 g/L CaCO₃, 100 mg/L ampicillin and 30 mg/Lkanamycin at a pressure of 0.1 atmosphere CO₂, at 30° C. at pH 3.5.

The GABA concentration is measured by high-performance liquidchromatography (HPLC), using an OptimaPak C18 column (4.6×150 mm, RSTech Corporation, Daejeon, Korea). The samples are centrifuged at 12,000rpm for 5 minutes, 100 μL of the supernatant transferred into a newEppendorf tube. The following reagents are added to these tubes: 200 μLof 1 M sodium bicarbonate buffer (pH 9.8), 100 μL of 80 g/L dansylchloride in acetonitrile and 600 μL of double-distilled water. Themixture is incubated at 80° C. for 40 minutes. The reaction is stoppedby adding 100 μL of 2% acetic acid. The mixture is centrifuged at 12,000rpm for 5 minutes. The supernatant is then filtered through a 0.2 μmMillipore filter and analyzed by HPLC on an Agilent system using a UVdetector. Derivatized samples are separated using a binary non-lineargradient using eluent A [tetrahydrofuran/methanol/sodium acetate 50 mMat pH 6.2 (5: 75: 420, by volume)] and eluent B (methanol). Residualglucose is measured with a bioanalyzer (YSI Inc.).

The carbon yield Y_(p/s) is calculated in grams of GABA produced pergram of glucose consumed.

This yield increases significantly by 15% for strain EQ.EC 020 ΔsucAΔzwf ΔgapA (RuBisCO+PRK)+(GadB) compared with the control strains EQ.EC013 ΔsucA (GadB).

Example 7: Improvement of Succinate and Oxalate Production in E. coli

An Escherichia coli K-12 strain, genetically modified to allowconstitutive expression of a glyoxylate dehydrogenase FPGLOXDH1 (GeneID: 946058) from Fomitopsis palustris, to reduce expression of the icdgene (Gene ID: 945702), and to inactivate the aceB (GeneID 948512) andsdhA (Gene ID: 945402) genes, would increase succinate and oxalic acidproduction yield.

The reduction in isocitrate dehydrogenase (icd) expression allows themetabolic flux to be redirected to the glyoxylic shunt. Inactivation ofmalate synthase (aceB) and succinate dehydrogenase (sdhA) prevents theglyoxylate and succinate, respectively, produced from being re-consumed.Deletion of the succinate dehydrogenase gene increases succinateproduction under aerobic conditions (Yang et al., Microbiol res. 2014May-June; 169(5-6):432-40). Deletion of the malate synthase gene allowsthe accumulation of glyoxylate which will be converted to oxalate by theconstitutive expression of glyoxylate dehydrogenase.

In this example, an Escherichia coli K-12 strain MG1655 in which thesdhA gene has been deleted is used. This strain is derived from a genedeletion bank (Baba et al. Mol Syst Biol. 2006; 2:2006.0008) inEscherichia coli K-12 and supplied by the coli Genetic Stock Centerunder the name JW0715-2 and with reference 8302. (JW0713-1: MG1655ΔsdhA::Kan).

a) Removal of the Selection Cassette by Specific Recombination of FTRRegions by Flp Recombinase

In order to be able to reuse the same deletion strategy as that used toconstruct strain JW0715-2 above (Rodriguez et al., 2016), the selectioncassette is deleted using a recombinase.

Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit by Gene Bridges) is transformed byelectroporation according to the kit protocol. The cells are selected onLB agar supplemented with 0.2% glucose, 0.0003% tetracycline and addedwith 0.3% L-arabinose. A counter-selection of the clones obtained iscarried out by verifying that they are no longer able to grow on thesame medium supplemented with 0.0015% kanamycin.

The strain obtained is called EQ.EC040: MG1655 ΔsdhA

b) Deletion of the aceB Gene

The deletion of the aceB gene (GeneID 948512) is performed by homologousrecombination and the use of the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit (Gene Bridges) according to the supplier'sprotocol.

Oligonucleotides designed to amplify an FRT-PKG-gb2-neo-FRT resistancegene expression cassette and having a 5′ sequence homologous over 50nucleotides to the adjacent regions of the deletion locus, i.e. atpositions 4215428 to 4215478.and 4217129.to 4217079 on the chromosomethus generating recombination arms of the cassette on the bacterialgenome on either side of the aceB gene coding sequence.

The Escherichia coli K-12 strain EQ.EC040 is transformed byelectroporation with plasmid pRedET according to the kit protocol. Thecolonies obtained are selected on rich complex medium LB agar with 0.2%glucose, 0.0003% tetracycline.

Transformation of the amplicon obtained in the first step in thepresence of RedET recombinase, induced by 0.3% arabinose in liquid LBfor 1 h. To that end, a second transformation of the deletion cassetteis performed by electroporation in cells expressing RedET and thecolonies are selected on LB agar supplemented with 0.2% glucose, 0.0003%tetracycline and added with 0.3% L-arabinose and 0.0015% kanamycin.

Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit by Gene Bridges) is transformed byelectroporation according to the kit protocol. The cells are selected onLB agar supplemented with 0.2% glucose, 0.0003% tetracycline and addedwith 0.3% L-arabinose. A counter-selection of the clones obtained iscarried out by verifying that they are no longer able to grow on thesame medium supplemented with 0.0015% kanamycin.

The strain obtained is called EQ.EC041: MG1655 ΔsdhA ΔaceB

c) Change in the icd Gene Promoter

i. Strategy

The replacement of the native promoter of the icd gene (Gene ID: 945702)by a weaker promoter is performed by homologous recombination and theuse of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit(Gene Bridges) according to the supplier's protocol.

ii. Introduction of the Weak Promoter P oxb1

The icd gene promoter is replaced by a cassette coupling the promoterP_(oxb1), characterized as weak, and an antibiotic resistance genecassette to allow the selection of the insertion of the P_(oxb1)cassette with an antibiotic resistance gene.

Oligonucleotides designed to amplify an FRT-PKG-gb2-neo-FRT resistancegene expression cassette and having a 5′ sequence homologous over 50nucleotides to the left adjacent region of the P_(icd) promoter locus(Genomic target LA) for the sense oligo, i.e. at positions 1194911 to1194961 on the genome, and the Spacer R sequence (Table 23) for thereverse oligo allow amplification of a fragment allowing assembly withthe P_(oxb1) fragment.

Oligonucleotides designed to amplify the P_(oxb1) promoter from plasmidPSF-OXB1 (Sigma # OGS553) and having a 5′ sequence homologous over 50nucleotides to the right adjacent region of the P_(icd) promoter locus(Genomic target RA) for the reverse oligo, i.e. at positions 1195173 to1195123 on the genome, and the Spacer S sequence (Table 25) for theoligo produce amplification of the P_(oxb1) fragment.

The amplification of a fusion fragment using the NEBuilder® HiFi DNAAssembly Master Mix Kit (E2321) allows the replacement promoter to becombined with an antibiotic selection cassette.

TABLE 25 Primer sequences for amplifying the OXB1 gene promoterSequences of homology Name with vector PSF-OXB1 POXB1-STCGTTGCGTTACACACAC (SEQ ID NO: 15) POXB1-R TGTGTCGAGTGGATGGTAG(SEQ ID NO: 16) Spacer S GCATGAATTCG (SEQ ID NO: 17) Spacer RCGAATTCATGC (SEQ ID NO: 18)

The Escherichia coli K-12 strain EQ.EC041 is transformed byelectroporation with plasmid pRedET according to the kit protocol. Thecolonies obtained are selected on rich complex medium LB agar with 0.2%glucose, 0.0003% tetracycline.

Transformation of the amplicon obtained in the first step in thepresence of RedET recombinase, induced by 0.3% arabinose in liquid LBfor 1 hour. To that end, a second transformation of the deletioncassette is performed by electroporation in cells expressing RedET andthe colonies are selected on LB agar supplemented with 0.2% glucose,0.0003% tetracycline and added with 0.3% L-arabinose and 0.0015%kanamycin.

Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit by Gene Bridges) is transformed byelectroporation according to the kit protocol. The cells are selected onLB agar supplemented with 0.2% glucose, 0.0003% tetracycline and addedwith 0.3% L-arabinose. A counter-selection of the clones obtained iscarried out by verifying that they are no longer able to grow on thesame medium supplemented with 0.0015% kanamycin.

The strain obtained is called EQ.EC042: MG1655 ΔsdhA ΔaceBP_(icd)::P_(oxb1)

d) Deletion of the zwf Gene

The deletion of the zwf gene (GeneID: 946370) is performed by homologousrecombination and the use of the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit (Gene Bridges) according to the supplier'sprotocol.

Oligonucleotides designed to amplify an FRT-PKG-gb2-neo-FRT resistancegene expression cassette and having a 5′ sequence homologous over 50nucleotides to the adjacent regions of the deletion locus, i.e. atpositions 1934789 to 1934839 and 1936364 to 1936314 on the chromosomethus generating recombination arms of the cassette on the bacterialgenome on either side of the entire operon.

The Escherichia coli K-12 strain EQ.EC042 is transformed byelectroporation with plasmid pRedET according to the kit protocol. Thecolonies obtained are selected on rich complex medium LB agar with 0.2%glucose, 0.0003% tetracycline.

Transformation of the amplicon obtained in the first step in thepresence of RedET recombinase, induced by 0.3% arabinose in liquid LBfor 1 h. To that end, a second transformation of the deletion cassetteis performed by electroporation in cells expressing RedET and thecolonies are selected on LB agar supplemented with 0.2% glucose, 0.0003%tetracycline and added with 0.3% L-arabinose and 0.0015% kanamycin.

Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit by Gene Bridges) is transformed byelectroporation according to the kit protocol. The cells are selected onLB agar supplemented with 0.2% glucose, 0.0003% tetracycline and addedwith 0.3% L-arabinose. A counter-selection of the clones obtained iscarried out by verifying that they are no longer able to grow on thesame medium supplemented with 0.0015% kanamycin.

The strain obtained is called EQ.EC043: MG1655 ΔsdhA ΔaceBP_(icd)::P_(oxb1) Δzwf

e) Deletion of the gapA Gene

The deletion of the gapA gene is performed by homologous recombinationand the use of the Quick & Easy E. coli Gene Deletion Red®/ET®Recombination Kit (Gene Bridges) according to the supplier's protocol.

Oligonucleotides designed to amplify an FRT-PKG-gb2-neo-FRT resistancegene expression cassette and having a 5′ sequence homologous over 50nucleotides to the adjacent regions of the deletion locus, i.e. thecoding phase of the gene (gapA) (GenBank: X02662.1) thus generatingrecombination arms of the cassette on the bacterial genome.

The Escherichia coli K-12 strain EQ.EC043 is transformed byelectroporation with plasmid pRedET according to the kit protocol. Thecolonies obtained are selected on rich complex medium LB agar with 0.2%glucose, 0.0003% tetracycline.

Transformation of the amplicon obtained in the first step in thepresence of RedET recombinase is induced by 0.3% arabinose in liquid LBfor 1 h. To that end, a second electroporation of the cells expressingRedET by the deletion cassette is performed and the colonies areselected on LB agar supplemented with 0.2% glycerol and 0.3% pyruvate,0.0003% tetracycline and added with 0.3% L-arabinose and 0.0015%kanamycin.

Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene DeletionRed®/ET® Recombination Kit by Gene Bridges) is transformed byelectroporation according to the kit protocol. The cells are selected onLB agar supplemented with 0.2% glucose, 0.0003% tetracycline and addedwith 0.3% L-arabinose. A counter-selection of the clones obtained iscarried out by verifying that they are no longer able to grow on thesame medium supplemented with 0.0015% kanamycin.

The strain obtained is called EQ.EC044: MG1655 ΔsdhA ΔaceBP_(icd)::P_(oxb1) Δzwf ΔgapA

f) Constitutive Overexpression of the FPGLOXDH1 and aceA Genes

The coding sequences (CDS) of the FPGLOXDH1 (Gene ID: 946058) and aceA(Gene ID: 948517) genes subcloned into a bacterial expression vectorpZE21MCS (EXPRESSYS) as synthetic operons according to the structuredescribed in Table 24. This vector has a ColE1 origin of replication anda kanamycin antibiotic resistance gene.

Each of these primers is coupled to floating sequences homologous over18 nucleotides at the ends of the fragment obtained by amplifying vectorpZE21MCS excluding the multiple cloning site. The two amplicons arecombined according to the protocol of the In-Fusion® HD Cloning Kit UserManual—Clontech to form plasmid pEQEC035 allowing the constitutiveoverexpression of the FPGLOXDH1 and aceA genes.

g) Insertion of the Engineering Required for CO₂ Fixation

For the recombinant expression of the different components of afunctional type I RuBisCO in E. coli, the genes described in Table 17(Example 4A]), are cloned in the form of a synthetic operon.

The coding sequences (CDS) of the genes described in the Table 2 areamplified and assembled into blocks according to the protocol providedwith the NEBuilder® HiFi DNA Assembly Master Mix Kit (E2321) to obtainthree integration blocks described in Table 26. Each block is thenamplified according to the protocol of the In-Fusion® HD Cloning KitUser Manual—Clontech to form the plasmids described below in Table 24.

TABLE 26 Composition of expression cassettes Structure of the syntheticoperon Block I Block II Block III Plasmid Vector type CDS A RBS1 CDS BRBS2 CDS C RBS3 CDS D pZA11 pZA11 pEQEC006 pZA11 rbcS D rbcL B rbcX Fprk pZE21MCS pZE21MCS pEQEC035 pZE21MCS FPGLOXDH1 D aceA

To control the expression level of these genes, ribosome bindingsequences (RBS) presented in Table 19 (Example 4B]), with variabletranslation efficiencies (Levin-Karp et al., ACS Synth Biol. 2013 Jun.21; 2(6):327-36. doi: 10.1021/sb400002n; Zelcbuch et al., Nucleic AcidsRes. 2013 May; 41(9):e98) are inserted between the coding phase for eachgene. The succession of each coding phase interspersed by an RBSsequence is constructed by successive insertions into a pZA11 vector(Expressys) that contains a PLtetO-1 promoter, a p15A origin ofreplication and an ampicillin resistance gene.

Several strains are produced by electroporating the different vectorspresented according to the plan below

EQ.EC045→(EQ.EC042+pZA11+pZE21MCS): MG1655 ΔsdhA ΔaceB P_(icd)::P_(oxb1)

EQ.EC046→(EQ.EC045+pEQEC006+pEQEC035): MG1655 ΔsdhA ΔaceBP_(icd)::P_(oxb1) Δzwf ΔgapA+(FPGLOXDH1+aceA)+(RuBisCO+PRK)

After transformation, clones are selected on LB glycerol, pyruvatemedium supplemented with 100 mg/L ampicillin and 30 mg/L kanamycin. Anadaptation and evolution phase of the strains with PRK and RuBisCOengineering is performed as described in Example 4A].

h) Production of Succinate and Oxalate

For the production of succinate and oxalate, cells from 500 mL of LBculture are inoculated into 20 mL of MS medium (40 g/L glucose, 1 g/LMgSO₄.7H₂O, 20 g/L (NH₄)₂SO₄, 1 g/L KH₂PO₄, 10 mg/L FeSO₄.7H₂O, 10 mg/LMnSO₄.7H₂O, 2 g/L yeast extract, 30 g/L CaCO₃, 100 mg/L ampicillin and30 mg/L kanamycin at a pressure of 0.1 atmosphere CO₂, at 30° C. at pH3.5.

The succinate concentration is measured by high-performance liquidchromatography (HPLC), culture samples are centrifuged at 12,000 g for 5min.

i. Succinate Determination

The culture supernatant is filtered through a 0.2 μm Millipore filterand analyzed on an Agilent HPLC system (series 1100) equipped with acation-exchange column. (Aminex HPX87-H, Bio-Rad, Hercules, Calif.,USA), a UV absorbance detector (Agilent Technologies, G1315D) and arefractive index (RI) detector (Agilent Technologies, HP1047A). Thesamples are separated on a 5 mM H₂S0₄ mobile phase at a flow rate of 0.4mL/min. The column oven temperature is 65° C.

Residual glucose is measured with a bioanalyzer (Ysi Inc.) or byHPLC-refractometry with an Aminex HPX87-H column.

The carbon yield Y_(p/s) is calculated in grams of succinate producedper gram of glucose consumed.

This yield increases significantly by 6% for the engineering strainEQ.EC046 compared with the control strain EQ.EC045 (empty).

ii) Oxalate Determination

The pellets are washed twice with 10 mM potassium phosphate buffer (pH7.5) containing 2 mM EDTA and stored at −20° C. Samples (1 mL) aretransferred into a tube pre-cooled with 0.75 g of glass beads (425-600μm) and introduced into a Fast Prep homogenizer (Thermo Scientific,Erembodegem, Netherlands) and subjected to 4 bursts of 20 s at speedcontrol 6. The lysates are centrifuged for 20 min at 4° C. and 36,000 g.Total protein determinations are performed according to the Lowry method(Lowry et al., 1951). Oxaloacetate acetyl hydrolase (EC 3.7.1.1.1)activity is measured using a modification of the direct opticaldetermination of oxaloacetate (OAA) at 255 nm as described in (Lenz etal., 1976). The disappearance of the OAA enol tautomer is checked at 255nm at 25° C. in a Hitachi Model 100-60 spectrophotometer (Hitachi,Tokyo, Japan), using quartz cuvettes. The 1 mL reaction mixture contains100 mM imidazole-HCl (pH 7.5), 0.9 mM MnCl₂.2H₂O, 1 mM OAA, 20 μL cellextract (controls with different volumes of cell extracts confirm thelinear relationship between enzyme activity and the amount of cellextract). The reaction is started by adding the cell extract.

The carbon yield Y_(p/s) is calculated in grams of doxalate produced pergram of glucose consumed.

This efficiency increases significantly by 3% for the engineering strainEQ.EC046 compared with the control strain EQ.EC045 (empty).

Example 8: Improvement of Citrate Production in Aspergillus niger

a) Strategy

The inactivation of the pgkA gene (Locus tag An08g02260), leading to thenon-functionality of the glycolysis pathway, and that of the gsdA gene(Locus tag An02g12140), inhibiting the oxidative part of the pentosephosphate pathway, are used to integrate the six genes for thefunctional expression of the PRK and RuBisCO enzymes, namely RbcS, RbcL,RbcX, GroES, GroEL and PRK for CO₂ fixation.

b) DNA Constructs

i) Guide RNA Sequences for Targeting the Gene to be Inactivated

In each of these two genes, a sequence of 20 nucleotides punctuated byan NGG motif (CRISPR target sequence underlined) was determined (Table27). In both cases, this sequence is specific to the targeted gene butalso unique in the Aspergillus niger genome. These sequences are used toexpress a guide RNA (gRNA) which, by forming a heteroduplex with thehomologous region of the Aspergillus niger genome, directs the action ofthe CAS9 endonuclease to induce a double-stranded break specifically onthe chosen locus.

TABLE 27 gRNA target sequence Reference Locus CRISPR Locus Gene genometag sequences 1 pgkA A. niger An08g CAACAAGGCCACTGG CBS 02260) TGGCCAGG513-88 (SEQ ID NO: 19) 2 gsdA A. niger An02g CATTTCCGGTCAATA CBS 12140)TGACAAGG 513-88 (SEQ ID NO: 20)

Plasmid pFC332 (Addgene #87845) described in Sarkari et al. (BioresourTechnol. 2017 December; 245(Pt B):1327-1333) contains a gRNA expressioncassette, a cassette allowing the functional expression of the Cas9endonuclease and an Hph cassette allowing the selection of this plasmid.The plasmid also contains the fragment AMA1_2.8 which allows transientpropagation of the plasmid. Finally, an origin of replication for E.coli is also present.

In order to target another gene, the gRNA cassette between FS A and FS Bcan be easily exchanged. This plasmid is modified by amplifying thedifferent parts of this plasmid, in order to eliminate the antibioticselection cassette and modify the 20 nucleotides allowing thespecificity of gRNA in favor of the sequences described in Table 27 toform plasmids pEQ0610 to target pgkA and pEQ0611 to target gsdA.

Donor Plasmid

Regions of Homologies with the Genome

The donor plasmid consists of an In-Fusion® HD Cloning Kit UserManual—Clontech assembly between plasmid pUC19 (GenBank: M77789.2) andthe genomic targeting sequences (LA and RA) of approximately 1500 bpeach, homologous to the locus chosen for integration. The LA and RAsequences are adjacent at 5′ and 3′ respectively to the locus sequencetargeted by the guide RNA. The genomic DNA/guide RNA heterodimer isrecognized by the Cas9 endonuclease for double-stranded cleavage (locus1: pgkA; locus 2: gsdA) (Table 28). The RA and LA fragments areamplified with primers for the pgkA gene and the gsdA gene (Table 29).The amplicon sequences are given in the sequence listing (SEQ ID NO: 55to SEQ ID NO: 58). An extension of 18 nucleotides on all forward primersof the three fragments is added according to the protocol of theIn-Fusion® HD Cloning Kit User Manual—Clontech, to allow a functionalassembly of the plasmids (pEQ0600 or pEQ0601) and the introduction oftwo restriction sites for type II restriction endonucleases (restrictionenzymes I-CeuI and I-Sce)I which have large asymmetric recognition sites(12 to 40 base pairs). These are recognition sequences of 18 base pairs,so rare. The fact that the cleavage is asymmetric at the reconnaissancesite allows the release of a fragment lacking sequences from bacterialvector pUC19. These two enzymes allow the integration block to beextracted by restriction after amplification by cloning in E. coli.

TABLE 28 Amplification of regions of homologies for the pgkA gene PrimerAmplicon Alias position Primer sequence 5′ pgkA_ LA1 ForwardGGATCGCAGATACGG A. niger TCGC (SEQ ID NO: 21) Reverse CCTCGGTGAAGACAACGCTG (SEQ ID NO: 22) 3′ pgkA_ RA1 Forward CTCCTTGAGAACCTG A. nigerCGTTTCC (SEQ ID NO: 23) Reverse CTGAAGTACGTTTTC CCAAGCC (SEQ ID NO: 24)

TABLE 29 Amplification of regions of homologies for the gsdA gene PrimerAmplicon Alias position Primer sequence 5′ gsdA_ LA2 ForwardCGTTATCACAAAGAA A. niger GCCAGGTCC (SEQ ID NO: 25) ReverseGCTGCTCTTCGATTT CCTTGGT (SEQ ID NO: 26) 3′ gsdA_ RA2 ForwardTCATCAACCTCAACA A. niger AGCACCTC (SEQ ID NO: 27) ReverseGTGAAGACAGCGGCG GTCC (SEQ ID NO: 28)

Engineering Expression Cassettes

The promoters and terminators are identified on the basis of GenBankdata. The selected promoters are determined from the +1 transcriptionpoint and go up 1.4 kb upstream in order to cover both the “core”sequences (TATA box) and the trans-activating sequences allowing theoptimal functionality of the promoter concerned.

For the terminators, the cut-off is made 500 bp after the stop codon ofthe gene.

The structure of each integration block of four expression cassettes isdefined as follows: the first level consists of simple elements, namelypromoters, coding sequences (CDS) and terminators. The promoter (Table30) and terminator (Table 31) elements, whose sequences are provided inthe sequence listing (SEQ ID NO: 59 to 62), are amplified and assembledwith the engineering CDS according to Table 32. The CDS, whose sequencesare provided in the sequence listing (SEQ ID NO: 63 to 66), areamplified according to the protocol provided with the NEBuilder® HiFiDNA Assembly Master Mix Kit (E2321) to obtain the functional expressioncassettes compiled in the table. Each integration block of four genes isorganized to include four different pairs (promoter/terminator) in orderto limit trans interference. Each integration block of six genes isorganized to include six different pairs (promoter/terminator) in orderto limit transcriptional interference

Donor Fragment for Insertion into the Target Locus of the Genome

The different multiple expression cassettes (RbcS, RbcL and RbcX) or(GroES, GroEL and PRK) are amplified and assembled around an antibioticselection cassette (Table), according to the protocol of the In-Fusion®HD Cloning Kit User Manual—Clontech, to form donor plasmids (pEQ0602 orpEQ0603).

TABLE 30 Native location of Aspergillus niger promotersused in genomic combinatorics to insert the sixgenes of the CO₂ fixation engineering into the Aspergillus niger genome.Pro- Gene Reverse Forward moters Organism ID primer primer PmbfAA. niger An02g TTTGAAGATGGA GCCATGAAATC CBS 12390 TGAGAAGTCGGCAATCATTTCC 513-88 (SEQ ID (SEQ ID NO: 33) NO: 29) PcoxA A. niger An07gTGTCCTGGTGGG GACGGCATTTG CBS 07390 TGGGTTG AGCAACATC 513-88 (SEQ ID(SEQ ID NO: 34) NO: 30) PsrpB A. niger An16g CTCGAACGAGAA TTGGCAGGGTCCBS 08910 TGGGAACC ACGTAGCC 513-88 (SEQ ID (SEQ ID NO: 35) NO: 31) PtvdAA. niger An04g GGCGGAATGAGA TTAGTCCATTC CBS 01530 TGCGACAG AGCAAGCTGCC513-88 (SEQ ID (SEQ ID NO: 36) NO: 32)

TABLE 31 Native location of Aspergillus Niger terminatorsused in genomic combinatorics to insert the sixgenes of the CO₂ fixation engineering into the Aspergillus niger genome.Ter- Gene Forward Reverse minators Organism ID primer primer TtrpC A.AN0648 TGATTTAATA GGGTAAACG nidulans GCTCCATGTC ACTCATAGG FGSC A4 AACAAGAGA (SEQ ID (SEQ ID NO: 37) NO: 41) TniaD A. AN1006 ACGGGTTCGC GGGATATTTnidulans ATAGGTTTGG GACACGATT FGSC A4 (SEQ ID CTGAGG NO: 38) (SEQ IDNO: 42) TgiaA A. niger An03g CGACCGCGAC CCGGAGATC CBS 513- 06550GGTGACTGAC CTGATCATC 88 (SEQ ID CG NO: 39) (SEQ ID NO: 43) TgpdAA. niger An16g GAATCAGGAC CGTGGTCTA CBS 513- 01830 GGCAAACTGA GCTGCCCTC88 AT C (SEQ ID (SEQ ID NO: 40) NO: 44)

TABLE 32 Assembly of expression cassettes Codon Expression optimi-Termi- cassette Gene GenBank zation Promoter nator CAS 1 RbcL BAD78320.1Yes PmbfA_(p) trpct CAS 2 RbcS BAD78319.1 Yes PcoxA_(p) TniaD CAS 3 RbcXBAD80711.1 Yes PsrpB_(p) glaAt CAS 4 Hph pUG75(P30671) No picdA_(p)TgpdA CAS 5 GroES U00096 No PmbfA_(p) trpct CAS 6 GroEL AP009048 NoPcoxA_(p) TniaD CAS 7 PRK BAD78757.1 Yes PsrpB_(p) glaAt CAS 8 BlepUG66(P30116) No picdA_(p) TgpdA

TABLE 33 Plasmid assembly Genomic Genomic Selection Plasmid sequencePromoter Gene Terminator sequence ori marker pEQ0600 LA1 RA1 coliAmpicillin pEQ0601 LA2 RA2 coli Ampicillin pEQ0602 LA2 PmbfA_(p) RbcLtrpct RA2 coli Ampicillin and PcoxA_(p) RbcS TniaD coli hydromycin BPsrpB_(p) RbcX glaAt coli picdA_(p) Hph TgpdA coli pEQ0603 LA1 PmbfA_(p)GroES Trpct RA1 coli Ampicillin and PcoxA_(p) GroEL TniaD coli bleomycinPsrpBp PRK glaAt coli picdAp Blue TgpdA coli

c) Transformation of Aspergillus niger

The transformation of DNA in Aspergillus niger is constrained by thepresence of the fungal cell wall, and is extremely ineffective comparedwith yeast or Escherichia coli. Nevertheless, the transformation ofprotoplasts prepared from fungal hyphae or conidiospores to germinationby treatment with cell wall degrading enzymes such as the cocktailconsisting of Lysing Enzyme® from Trichoderma harzianum, chitinase fromStreptomyces griseus and β-glucuronidase from Helix pomatia (de Bekkeret al., J Microbiol Methods. 2009 March; 76(3):305-6) allowstransformants to be produced.

The A. niger strain CBS 513-88 is grown at 30° C. in a 1 L Erlenmeyerflask with 250 mL of transformation medium (Kusters-van Someren et al.,Curr Genet. 1991 September; 20(4):293-9). After growth for 16 h at 250rpm, the mycelium is collected by filtration on Miracloth (Calbiochem)and washed with deionized water. Protoplasts are prepared in thepresence of 5 g/L lysis enzymes from Trichoderma harzianum (Sigma SaintLouis, Mo., USA), 0.075 Uml-1 chitinase from Streptomyces griseus(Sigma) and 460 Uml-1 glucuronidase from Helix pomatia (Sigma) in KMC(0.7 M KCl, 50 mM CaCl₂, 20 mM Mes/NaOH, pH 5.8) for 2 hours at 37° C.and 120 rpm. Protoplasting is monitored every 30 minutes with amicroscope. The protoplasts are filtered through a Miracloth filter andcollected by centrifugation at 2000×g and 4° C. for 10 minutes. Theprotoplasts are washed with cold STC (1.2 M sorbitol, 10 mM Tris/HCl, 50mM CaCl₂, pH 7.5) and then resuspended in 100 pi of STC and useddirectly for the transformation.

In order to integrate a metabolic pathway into the A. niger genome,co-transformation of a plasmid and a linear fragment is required.Plasmid pEQ0610 is co-transformed with a donor fragment to integratepart of the engineering into the genome while inactivating the pgkAgene. Similarly, plasmid pEQ0611 is co-transformed with a donor fragmentto integrate the other part of the engineering into the genome whileinactivating the gsdA gene. These sequences serve both as matrices forhomologous recombination and as selection markers: during integrationwith functional expression of the antibiotic resistance genes Hph orBle. The strains are directly selected on minimal medium plates with anaddition of hygromycin B or bleomycin allowing direct selection on theintegration event. Due to the presence of the origin of replicationAMA1_2.8, plasmid pCAS_pyrG2 is easily lost causing only transientexpression of the Cas9 protein, thus reducing the risk of non-targetedadverse effects.

Linear cassettes (10 μg) and plasmid (5 μg) are mixed with 100 μL of STCsolution containing at least 10⁷ protoplasts and 330 μL of freshlyprepared polyethylene glycol (PEG) solution (25% PEG 6000, 50 mM CaCl₂,10 mM Tris/HCl, pH 7.5) and kept on ice for 20 minutes. After mixingwith an additional 2 mL PEG solution and incubating at room temperaturefor 10 minutes, the protoplast mixture is diluted with 4 mL of STC.

The selection of transformants is carried out on MM plates with 150μg/mL hygromycin B added or MM plates with 50 μg/mL bleomycin added. Alltransformants are purified by isolating single colonies from theselection medium at least twice. The insertion of the fragments isverified by sequencing the target locus with the appropriate controlprimers. Genomic DNA from fungal cells is isolated with a modifiedprotocol, using the Wizard® Genomic DNA Purification Kit (Promega,Wisconsin, USA). The mycelium is cultured overnight in CM (30° C., 150rpm) in 290 pi of 50 mM EDTA solution and 10 pi of lyticase (10 mg/mL)to remove the cell wall. After 90 minutes of incubation at 37° C., thesuspension is centrifuged and the supernatant is discarded. The myceliumpellet is resuspended in 300 μL of nuclei lysis solution and 100 μL ofprotein precipitation solution. The samples are incubated on ice for 5minutes and centrifuged. The DNA is precipitated with isopropanol andwashed with 70% ethanol. The DNA pellet is rehydrated with a DNArehydration solution containing RNase (100 μg/mL). The successfultransformation and integration of the expression cassettes was verifiedby PCR.

TABLE 34 Strains used for the yield study Strains Genome Geneticmodification EQ1500 A. niger CBS 513-88 EQ1501 A. niger gsdA ::PmbfA_(p)-RbcL-trpc; PcoxA_(p-)RbcS-TniaD; CBS picdA_(p)-Hph-TgpdA;PsrpB_(p)-RbcX-glaAt 513-88 EQ1502 A. niger gsdA :: PmbfA_(p)-RbcL-trpc;PcoxA_(p-)RbcS-TniaD; CBS picdA_(p)-Hph-TgpdA; PsrpB_(p)-RbcX-glaAt513-88 pgkA :: PmbfA_(p)-GrES-trpc; PcoxA_(p-)GroEL-TniaD;picdA_(p)-Ble-TgpdA; PsrpB_(p)-PRK-glaAt

Conidia (10⁸/L) from strains EQ1500 and EQ1502 are inoculated andcultured at 30° C. on a rotary shaker (180 rpm) in shaker flaskscontaining Vogel medium without MnSO₄ with a total glucose content of15% and a total nitrogen content of 0.2% and 10% CO₂. The determinationof glucose and organic acids was performed as described above (Blumhoffet al., 2013; Steiger et al., 2016) on an HPLC (Shimadzu, Kyoto; Japan)equipped with an Aminex HPX-87 H column (300×7.8 mm, Bio-Rad, Hercules,Calif.). A refractive index detector (RID-10 A, Shimadzu) is used forthe detection of glucose and citric acid, while a PDA detector(SPD-M20A, Shimadzu) at 300 nm is used to detect cis-aconitic andtrans-aconitic acid. The column is used at 60° C. at a flow rate of 0.6mL/min and with a 0.004 M H₂SO₄ aqueous solution as mobile phase. Theculture was carried out in three biological replicates.

d) Analytical Method

For the quantification of extracellular metabolites, a culture sample iscentrifuged at 14,000×g for 5 min. The supernatant is filtered through afilter with a 0.45 pm pore size. The filtrate is maintained at −20° C.until analysis. The concentration of citrate and of oxalate is detectedand quantified with ultraviolet light at 210 nm using an Amethyst C18-Hcolumn (250×4.6 mm, Sepax Technologies, Newark, Del., USA). Elution iscarried out at 30° C. with 0.03% H₃P0₄ at a flow rate of 0.8 mL/min.Reducing sugar is detected with the 3,5-dinitrosalicylic acid method.Biomass determination: 5 mL of sample is filtered through Miracloth(Calbiochem, San Diego, Calif., USA) to collect hyphae and washed withdistilled water. The hyphae are heated to 105° C. in a “Miracloth”. Forthe calculation of the dry cell weight (DCW), the weight of Miracloth ismeasured beforehand and subtracted from the total weight to give the netweight, then the net weight per unit volume is calculated as DCW.

After complete analysis, the comparison of citric acid production yieldas a function of glucose consumption is 18% higher in the engineeredstrain EQ1502 than in the wild-type strain EQ1500.

Example 9: Improvement of Itaconate Production in Aspergillus terreus

a) Strategy

Inactivation of the pgkA gene (Locus tag (ATEG_00224), leading to thenon-functionality of the glycolysis pathway, and that of the gsdA gene(Locus tag ATEG_01623), inhibiting the oxidative part of the phosphatepentose pathway, are used to integrate the six genes allowing thefunctional expression of the PRK and RuBisCO enzymes, namely rbcS, rbcL,rbcX, groES, groEL and prk allowing CO₂ fixation.

b) DNA Constructs

i) RNA Guide Sequences to Target the Gene to be Inactivated

In each of these two genes, a sequence of 20 nucleotides punctuated byan NGG motif (CRISPR target sequence underlined) was determined (Table35). In both cases, this sequence is specific to the targeted gene butalso unique in the Aspergillus terreus genome. These sequences are usedto express a guide RNA (gRNA) which, by forming a heteroduplex with thehomologous region of the Aspergillus terreus genome, directs the actionof the CAS9 endonuclesae to induce a double-stranded break specificallyon the selected locus. For pgkA, the sequence identified in the secondintron, the first 20 nucleotides have a unique pattern in the genome,even allowing two mismatches. For gsdA, the sequence identified in thefourth intron, the first 20 nucleotides have a unique pattern in thegenome, even allowing two mismatches.

TABLE 35 Guide RNA target sequence Reference Locus Locus Gene genome tagCRISPR sequences 3 pgkA A. (ATEG_ CTGCGTCGGCAA terreus 00224)GGAAGTTGAGG NIH262 (SEQ ID NO: 45) 4 gsdA A. (ATEG_ CATCAGCGGCCA terreus01623) ATATGACAAGG NIH2624 (SEQ ID NO: 46)

Plasmid pFC332 (Addgene #87845) described in Sakari et al. (Bioresourtechnol. 2017; 245(Pt B):1327-1333) contains a gRNA expression cassette,a cassette for the functional expression of the Cas9 endonuclease and anHph cassette for the selection of this plasmid. The plasmid alsocontains the fragment AMA1_2.8 which allows transient propagation of theplasmid. Finally, an origin of replication for E. coli is also present.

In order to target another gene, the gRNA cassette between FS A and FS Bcan be easily exchanged. Thus, this plasmid is modified by amplifyingthe different parts of this plasmid in order to eliminate the antibioticselection cassette and to modify the 20 nucleotides allowing thespecificity of gRNA in favor of the sequences described in Table 35 toform plasmids pEQ0615 to target pgkA and pEQ0616 to target gsdA in theAspergillus terreus genome.

ii) Donor Plasmid

Regions of Homology with the Genome

The donor plasmid consists of an In-Fusion® HD Cloning Kit UserManual—Clontech assembly between plasmid pUC19 (GenBank: M77789.2) andgenomic targeting sequences (LA and RA) of approximately 1500 bp eachhomologous to the locus chosen for integration. The LA and RA sequencesare adjacent at 5′ and 3′ respectively to the locus sequence targeted bythe guide RNA. The genomic DNA/guide RNA heterodimer is recognized bythe Cas9 endonuclease for double-stranded cleavage (locus 1: pgkA; locus2: gsdA) (Table 35). The RA and LA fragments are amplified with theprimers described in Table 36, for the pgkA gene, and Table 37, for thegsdA gene. The amplicon sequences are in the sequence listing (SEQ IDNO: 67 to 70).

An extension of 18 nucleotides on all forward primers of the threefragments is added according to the protocol of the In-Fusion® HDCloning Kit User Manual—Clontech to allow a functional assembly of theplasmids (pEQ0604 or pEQ0605) (33) and the introduction of tworestriction sites for type II restriction endonucleases (restrictionenzymes I-CeuI and I-SceI) which have large asymmetric recognition sites(12 to 40 base pairs). These are recognition sequences of 18 base pairs,therefore rare and not present in the described assembly. The fact thatthe cleavage is asymmetric at the reconnaissance site allows a fragmentdevoid of sequences to be released from the bacterial vector pUC19.These two enzymes allow the integration block to be extracted byrestriction after amplification by cloning in E. coli.

TABLE 36 Amplification of regions of homologies for the pgkA gene PrimerAmplicon position Primer sequence 5′ pgkA_ Forward CTTGGGGAATTGGAterreus GACACG (SEQ ID NO: 47) Reverse TCTTGCCGATGAG CTTCTCC(SEQ ID NO: 48) 3′ pgkA_ Forward CAGATCATCCTCC Aterreus TGGAGAACC(SEQ ID NO: 49) Reverse ACGGCACGAATGT TCACCTG (SEQ ID NO: 50)

TABLE 37 Amplification of regions of homologies for the gsdA gene PrimerAmplicon position Primer sequence 5′ gsdA_ Forward ATTGGAAGCTGGCTCTAttereus ATCTCACC (SEQ ID NO: 51) Reverse GCTGTTCTTCGATTTC CTTGGTG(SEQ ID NO: 52) 3′ gsdA_ Forward TCAACCTCACCAAGCA Aterreus CCTCG(SEQ ID NO: 53) Reverse CAAACAGCCCGTCGCA ACTG (SEQ ID NO: 54)

Engineering Expression Cassettes

Promoters and terminators are identified on the basis of GenBank data.The selected promoters are determined from the +1 transcription pointand go up 1.4 kb upstream in order to cover both the “core” sequences(TATA box) and the trans-activating sequences allowing the optimalfunctionality of the promoter concerned.

For the terminators, the cut-off is made 500 bp after the stop codon ofthe gene.

The structure of each integration block of four expression cassettes isdefined as follows: the first level consists of simple elements, namelypromoters, coding sequences (CDS) and terminators. The promoter (Table30) and terminator (Table 31) elements are amplified and assembled withthe engineering CDS according to Table 32. The CDS are amplifiedaccording to the protocol provided with the NEBuilder® HiFi DNA AssemblyMaster Mix Kit (E2321) in order to obtain the functional expressioncassettes compiled in the table. Each integration block of four genes isorganized to include four different terminator promoter pairs in orderto limit trans interference Each integration block of six genes isorganized to include six different terminator promoter pairs in order tolimit transcriptional interference.

Donor Fragment for Insertion into the Target Locus of the Genome

The different multiple expression cassettes (RbcS, RbcL and RbcX orGroES, GroEL and PRK are amplified and assembled around an antibioticselection cassette (Table 38), according to the protocol of theIn-Fusion® HD Cloning Kit User Manual—Clontech, to form donor plasmids(pEQ0606 or pEQ0607).

TABLE 38 Plasmid assembly Genomic Genomic Selection Plasmids sequencePromoter Gene Terminator sequence ori marker pEQ0604 LA4 RA4 coliAmpicillin pEQ0605 LA3 RA3 coli Ampicillin pEQ0606 LA4 PmbfA_(p) rbcLtrpct RA4 coli Ampicillin and PcoxA_(p) rbcS TniaD coli hydromycin BPsrpB_(p) rbcX glaAt coli picdA_(p) Hph TgpdA coli pEQ0607 LA3 PmbfA_(p)groES trpct RA3 coli Ampicillin and PcoxA_(p) groEL TniaD coli bleomycinPsrpB_(p) prk glaAt coli picdA_(p) Ble TgpdA coli

c) Transformation of Aspergillus terreus

The transformation of Aspergillus terreus DNA is carried out inaccordance with the strategy applied for Aspergillus niger (Example 8)using A. terreus strain NIH262.

TABLE 39 Strains used for the yield study Strains Genome Geneticmodification EQ1600 A. terreus NIH262 EQ1601 A. terreus gsdA ::PmbfA_(p)-RbcL-trpc; PcoxA_(p-)RbcS-TniaD; NIH262 picdA_(p)-Hph-TgpdA;PsrpB_(p)-RbcX-glaAt EQ1602 A. terreus gsdA :: PmbfA_(p)-RbcL-trpc;PcoxA_(p-)RbcS-TniaD; NIH262 picdA_(p)-Hph-TgpdA; PsrpB_(p)-RbcX-glaAtpgkA :: PmbfA_(p)-GrES-trpc; PcoxA_(p-)GroEL-TniaD; picdA_(p)-Ble-TgpdA;PsrpB_(p)-PRK-glaAt

Culture of A. terreus strains EQ1600 and EQ1602 on 3% glucose.

The optimized media composition described by Hevekerl et al. (ApplMicrobiol Biotechnol. 2014; 98:6983-6989) is used. It contains 0.8 gKH₂P0₄, 3 g NH₄N0₃, 1 g MgSO₄.7H20, 5 g CaCl₂.2 H₂0, 1.67 mg FeCl₃.6H₂O,8 mg ZnSO₄.7H₂O and 15 mg CuSO₄.7H2O per liter. To mimic the typicalsugar concentration obtained from wheat straw hydrolysate (150 g/L)pretreated with dilute acid (0.75% v/v, 160° C., 10 min) andenzymatically saccharified (pH 5.0, 45° C., 72 h), an adequate amount ofglucose up to 30 g/L is used. Sugars and all other components are addedfrom sterile stock solutions. The pH of the medium without CaCl₂ isadjusted to 3.1 with 0.5 M H₂SO₄ before inoculating the sporepreparation for strains EQ1600 and EQ1602. The culture is carried outunder shaking with 25 mL of medium in 125 mL Erlenmeyer flasks at 33° C.in a rotary shaker at 200 rpm for 7-10 days in an environment of 10%CO₂. The pH is not checked during fermentation. Shaking of the flasks ismaintained during sampling for time studies to ensure a continuoussupply of oxygen. All experiments are carried out in triplicate. Allmedia components are obtained from Sigma Chemical, St. Louis, Mo. Forthese experiments, each sugar was dissolved in deionized water andpassed through a column (440×45 mm) of Dowex 50-X8 (100/200 mesh)cation-exchange resin (Bio-Rad Laboratories, Hercules, Calif.) to removemanganese, if necessary.

d) Analytical Procedures

The concentration of the cell mass is determined from the dry cellweight. The cell mass present in the fermentation broth is collected bycentrifugation at 10,000 g for 10 minutes and carefully rinsed threetimes with deionized water. The rinsed cell mass was completely dried at80° C. until a constant weight was obtained. The fermentation brothafter centrifugation (10,000 g, 10 min) is stored at −20° C. beforeanalysis of glucose, itaconic acid and by-products (succinic acid,α-ketoglutaric acid, malic acid, cis-aconitic acid, and trans-aconiticacid) using high-performance liquid chromatography (HPLC). A ShimadzuProminence HPLC system (Shimadzu America, Inc., Columbia, Md.) is used.Two columns (Aminex HPX-87P column, 300×7.8 mm with ash removalcartridge and Carbo-P protection cartridge, and one Aminex HPX 87Hcolumn, 300×7.8 mm with Microguard Cation H cartridge (Bio-Rad)) areused for the analysis of sugars and organic acids, respectively. TheAminex HPX 87P column is maintained at 85° C. and glucose is eluted withMilli-Q acidified deionized water (Millipore, Bedford, Mass.) at a flowrate of 0.6 mL/min.

The Aminex HPX 87H column is maintained at 65° C. and sugars and organicacids are eluted with 5 mM H₂SO₄ prepared using Milli-Q deionizedfiltered water at a rate of 0.5 mL/min. Detection is carried out using arefractive index detector for sugars and a 210 nm UV detector fororganic acids. Propionic acid (1%, weight/volume) is used as internalstandard to estimate the liquid lost during aerobic fermentation for7-10 days at 33° C. under 10% CO₂. All HPLC standards, including organicacids, are purchased from Sigma. The manganese concentration (ppb level)is determined using an Optima 7000DV (Perkin-Elmer, Waltham, Mass.)inductively coupled plasma optical emission spectrometer (ICP-OES) bythe procedure described by Bakota et al. (Eur J Lipid Sci Technol. 2015;117:1452-1462.

Based on the results of the production of itaconic acid from glucose, amass yield increment of itaconic acid from glucose of 15% is observedfor the engineered strain EQ1602 compared with the reference strainEQ1600.

1. A genetically modified microorganism for the production of anexogenous molecule of interest and/or to overproduce an endogenousmolecule of interest, other than a RuBisCO or phosphoribulokinaseenzyme, said microorganism expressing a functional RuBisCO enzyme and afunctional phosphoribulokinase (PRK), and in which the glycolysispathway is at least partially inhibited, upstream of the production of1,3-biphospho-D-glycerate (1,3-BPG) or upstream of the production of3-phosphoglycerate (3PG), and downstream of the production ofglyceraldehyde-3-phosphate (G3P), wherein said microorganism isgenetically modified so as to produce the exogenous molecule of interestand/or to overproduce the endogenous molecule of interest, other than aRuBisCO or phosphoribulokinase enzyme.
 2. The genetically modifiedmicroorganism according to claim 1, wherein the oxidative branch of thepentose phosphate pathway is also at least partially inhibited.
 3. Thegenetically modified microorganism according to claim 1, wherein saidmicroorganism is genetically modified to express a recombinant RuBisCOenzyme and/or PRK.
 4. The genetically modified microorganism accordingto claim 2, wherein said microorganism is genetically modified toinhibit the oxidative branch of the pentose phosphate pathway upstreamof ribulose-5-phosphate production.
 5. The genetically modifiedmicroorganism according to claim 1, wherein the exogenous moleculeand/or the endogenous molecule is selected from amino acids, peptides,proteins, vitamins, sterols, flavonoids, terpenes, terpenoids, fattyacids, polyols and organic acids.
 6. The genetically modifiedmicroorganism according to claim 1, wherein said microorganism is aeukaryotic cell or a prokaryotic cell.
 7. The genetically modifiedmicroorganism according to claim 1, wherein the expression of the geneencoding glyceraldehyde 3-phosphate dehydrogenase is at least partiallyinhibited.
 8. The genetically modified microorganism according to claim1, wherein the expression of the gene encoding phosphoglycerate kinaseis at least partially inhibited.
 9. The genetically modifiedmicroorganism according to claim 7, wherein the expression of the geneencoding glucose-6-phosphate dehydrogenase or 6-phosphogluconolactonaseor 6-phosphogluconate dehydrogenase is at least partially inhibited. 10.The genetically modified microorganism according to claim 1, whereinsaid microorganism is a yeast of the genus Saccharomyces cerevisiaegenetically modified to express a functional type I or II RuBisCO and afunctional phosphoribulokinase (PRK), and in which the expression of theTDH1, TDH2 and/or TDH3 gene is at least partially inhibited.
 11. Thegenetically modified microorganism according to claim 1, wherein saidmicroorganism is a Saccharomyces cerevisiae yeast genetically modifiedto express a functional type I or II RuBisCO and a functionalphosphoribulokinase (PRK), and in which the expression of the PGK1 geneis at least partially inhibited.
 12. The genetically modifiedmicroorganism according to claim 10, wherein the expression of the ZWF1gene is at least partially inhibited.
 13. The genetically modifiedmicroorganism according to claim 1, wherein said microorganism is afilamentous fungus of the genus Aspergillus genetically modified toexpress a functional type I or II RuBisCO and a functionalphosphoribulokinase (PRK), and in which the expression of the pgk andgsdA genes is at least partially inhibited.
 14. The genetically modifiedmicroorganism according to claim 1, wherein said microorganism is an E.coli bacterium genetically modified to express a functional type I or IIRuBisCO and a functional phosphoribulokinase (PRK), and in which theexpression of the gapA and/or pgk gene, and optionally the zwf gene, isat least partially inhibited. 15.-16. (canceled)
 17. A biotechnologicalprocess for producing at least one molecule of interest, wherein itcomprises a step of culturing a genetically modified microorganism asdefined in claim 1, under conditions allowing the synthesis orbioconversion by said microorganism of said molecule of interest, andoptionally a step of recovering and/or purifying said molecule ofinterest.
 18. The biotechnological process according to claim 17,wherein the molecule of interest is selected from amino acids, peptides,proteins, vitamins, sterols, flavonoids, terpenes, terpenoids, fattyacids, polyols and organic acids.
 19. The biotechnological processaccording to claim 17, wherein the molecule of interest is selectedglutamate, citrate, itaconate or GABA.
 20. The biotechnological processaccording to claim 17, wherein the microorganism is genetically modifiedto express at least one enzyme involved in the bioconversion orsynthesis of said molecule of interest.
 21. The biotechnological processaccording to claim 17, wherein the microorganism is genetically modifiedto at least partially inhibit an enzyme involved in the degradation ofsaid molecule of interest.
 22. A process for producing a molecule ofinterest comprising (i) inserting at least one sequence encoding anenzyme involved in the synthesis or bioconversion of said molecule ofinterest into a recombinant microorganism as defined in claim 1, (ii)culturing said microorganism under conditions allowing expression ofsaid enzyme and optionally (iii) recovering and/or purifying saidmolecule of interest.
 23. A process for producing a molecule of interestcomprising (i) inhibiting the expression of at least one gene encodingan enzyme involved in the degradation of said molecule of interest in arecombinant microorganism as defined in claim 1, (ii) culturing saidmicroorganism under conditions allowing expression of said enzyme andoptionally (iii) recovering and/or purifying said molecule of interest.24. The genetically modified microorganism of claim 1, wherein saidmicroorganism is a eukaryotic cell selected from yeasts, fungi andmicroalgae.
 25. The genetically modified microorganism of claim 1,wherein said microorganism is a bacterium.